STRUCTURE AND FUNCTION OF TRYPTOPHAN HYDROXYLASE

By

GEORGE CHIH-THAI JIANG

A Thesis Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE in the Molecular Genetics Program of the Wake Forest University School of Medicine May 2003 Winston-Salem, North Carolina Approved by: Kent E. Vrana, Ph.D., Advisor _______________________ Examining Committee: Linda C. McPhail, Ph.D., Chairman _______________________ Mark O. Lively, Ph.D. _______________________ Leslie B. Poole, Ph.D. _______________________ William E. Sonntag, Ph.D. _______________________

DEDICATION

I would like to dedicate this work to my parents, Allan Lii-Shang and Grace

Mei-Chuon Jiang. Without your constant love and support throughout the turbulent

times of my graduate career, this would not have been possible. Thank you for your

constant words of wisdom and encouragement.

ii

ACKNOWLEDGMENTS

First and foremost, I would like to thank Dr. Kent E. Vrana. I appreciate all

the patience and guidance to achieve my career goals. My time at Wake Forest

University has been filled with many ups and downs and I thank you for always

supporting me, taking the time to further motivate and guide me in the right direction,

and providing numerous opportunities for professional development.

I would also like to acknowledge Dr. James Smith and the entire Department

of Physiology and Pharmacology for providing the resources and supportive

environment that have made my graduate career here so enjoyable. I would like to

specifically thank all the members of the Vrana Lab, past and present, for their

assistance and friendship in the laboratory. In particular, I would like to thank Dr.

George J. Yohrling IV for his guidance and for serving as the model student-scientist

which I have aspired to emulate. I would also like to thank Amanda Domer for all

her assistance with protein expression, purification and enzymatic assays.

Special thanks to Dean Melson, Dean Solano, Pearl Lawrence, and Beth

Whitsett for their help in all aspects of the Graduate Student Association, and their

support and assistance throughout my graduate career. I would also like to thank

my committee members, Drs. Lively, McPhail, Poole, and Sonntag for their patience

and guidance through the scientific process. Finally, but not least, I would like to

thank all my classmates, friends, and colleagues. The support network you have

provided has been remarkable and is greatly appreciated.

iii

TABLE OF CONTENTS LIST OF FIGURES AND TABLES .......................................................................vi ABBREVIATIONS .............................................................................................. viii ABSTRACT.......................................................................................................... x CHAPTER I - INTRODUCTION: STRUCTURAL AND FUNCTIONAL

CHARACTERIZATION OF THE AROMATIC AMINO ACID HYDROXYLASES ..................................................................................... 1

Aromatic Amino Acid Hydroxylases...................................... 2 Tryptophan Hydroxylase and Serotonin Biosynthesis .......... 6 Serotonin in Human Health and Disease.............................. 7 AAAH Polymorphisms and Disease ..................................... 9 Protein Purification Studies ................................................ 13 X-Ray Crystallographic Studies.......................................... 17 References ......................................................................... 23 Statement of Purpose......................................................... 33

CHAPTER II - IDENTIFICATION OF SUBSTRATE ORIENTING AND

PHOSPHORYLATION SITES WITHIN TRYPTOPHAN HYDROXYLASE USING HOMOLOGY-BASED MOLECULAR MODELING ..................... 35

This chapter is published in Journal of Molecular Biology 302:1005-1017 (2000).

Abstract .............................................................................. 36 Introduction........................................................................ 38 Results .............................................................................. 44 Discussion ......................................................................... 60 Materials and Methods ....................................................... 71 References ........................................................................ 78

CHAPTER III – FUNCTIONAL ANALYSIS OF THE V177I CODING REGION

POLYMORPHISM IN HUMAN TRYPTOPHAN HYDROXYLASE (TPH1) 82 In Preparation, Journal of Neurochemistry (2003)

Abstract ............................................................................. 83 Introduction........................................................................ 84 Materials and Methods ...................................................... 88

iv

Results .............................................................................. 94 Discussion ......................................................................... 96 References ...................................................................... 107

CHAPTER IV - SUMMARY AND CONCLUSIONS ......................................... 113

References ...................................................................... 121 APPENDIX - PHENYLKETONURIA …………………………………………..….. 124

This chapter is published in Encyclopedia of Life Sciences 14:150-154 (2000).

Introduction...................................................................... 125 Clinical Signs and Symptoms ........................................... 126 Classical Phenylketonuria ............................................... 132 Nonclassical Phenylketonuria.......................................... 137 Future Treatments for Phenylketonuria ........................... 139 Summary ......................................................................... 143 Further Reading .............................................................. 144

CURRICULUM VITA ....................................................................................... 145

v

LIST OF FIGURES AND TABLES

CHAPTER I

Figure 1: Biosynthetic pathway for catecholamines and indoleamines........... 5 Table I: Published TPH purification protocols ............................................ 13 Figure 2: Structural comparisons of the AAAHs ........................................... 22

CHAPTER II Table 1: Published TPH mutagenesis studies............................................. 41 Figure 1: Hypothetical model of human TPH (monomer) ............................. 46 Figure 2: Side by side comparison of TH, PAH, and hypothetical TPH........ 48 Figure 3: Tetramer of hypothetical TPH ....................................................... 50 Table 2: Steady-state kinetic parameters of wild-type TPH and mutants.... 51 Figure 4: Phosphorylation and immunodetection of TPH ............................. 53 Figure 5: Specific activities of TPH active-site mutations ............................. 57 Figure 6: Lineweaver-Burk analysis of wild-type TPH and TPH Y235A ....... 59 Figure 7: Multiple sequence alignment ........................................................ 68 Figure 8: Sequence alignment of the aromatic amino acid hydroxylases..... 70

CHAPTER III Table I: Site-directed mutagenesis primers................................................ 90 Figure 1: Structural depictions of Val177 mutations ................................... 102

vi

Table II: Purification data for wild-type and mutant enzymes.................... 103 Figure 2: Coomassie and immunoblots of purified proteins........................ 105 Table III: Steady-state kinetic parameters of wild-type TPH and mutants.. 106

CHAPTER IV Figure 1: Structural location of the functional residues of interest in the

present work. .............................................................................. 118 APPENDIX Figure 1: Phenylalanine metabolism ......................................................... 128 Table 1: Genetic defects in phenylketonuria ............................................ 135

vii

ABBREVIATIONS

5-HT 5-Hydroxytryptamine (Serotonin)

5-HTP 5-Hydroxytryptophan

AAADC Aromatic Amino Acid Decarboxylase (EC 4.1.1.28)

AAAH Aromatic Amino Acid Hydroxylase(s)

BH4 Tetrahydrobiopterin ((6R)-5,6,7,8-Tetrahydro-L-biopterin)

CaMPKII Calcium/Calmodulin-Dependent Protein Kinase II

C∆/N∆ Carboxyl/Amino Terminal Deletion

cDNA Complementary DNA

CNS Central Nervous System

DA Dopamine (3-hydroxydopamine)

DNA Deoxyribonucleic Acid

DOPA 3,4-Dihydroxyphenylalanine

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic Acid

Fe Iron

HEPES N-[2-Hydroxyethyl] Piperazine-N’-[2-Ethanesulfonic Acid]

kb Kilobase

kDa Kilodalton

Km Michaelis-Menten Constant

mRNA Messenger Ribonucleic Acid

OCD Obsessive Compulsive Disorder

viii

PAH Phenylalanine Hydroxylase (phenylalanine-4-monooxygenase; EC

1.14.16.1)

PD Parkinson’s Disease

PKA Protein Kinase A; cAMP-Dependent Protein Kinase

PMSF Phenylmethylsulfonyl Fluoride

RMSD Root Mean Square Deviation

SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

SEC Size-Exclusion Chromatography

SN Substantia Nigra

SNP Single Nucleotide Polymorphisms

TH Tyrosine Hydroxylase (tyrosine-3-monooxygenase; EC 1.14.16.2)

TPH Tryptophan Hydroxylase (tryptophan-5-monooxygenase; EC

1.14.16.4)

Vmax Maximal Velocity

ix

ABSTRACT

George C.-T. Jiang

STRUCTURE AND FUNCTION OF TRYPTOPHAN HYDROXYLASE

Thesis under the direction of

Kent E. Vrana, Ph.D., Professor of Physiology and Pharmacology and Director of

Graduate Studies

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis

of serotonin, and is a member of a family of enzymes known as aromatic amino acid

hydroxylases (AAAH). Studies into the regulation, structure, and function of TPH

have lagged behind those of the other AAAH. In the absence of an experimentally-

determined crystal structure, we generated a hypothetical model of human TPH

using multiple sequence alignment homology-based molecular modeling of the other

AAAH. We then performed site-directed mutagenesis to identify functional domains

within the TPH protein, and to validate the hypothetical model. Our studies

demonstrated that tyrosine 235 plays a role in tryptophan substrate interactions in

the active site, and that serine 58 and serine 260 are substrates for Ca2+/calmodulin-

dependent protein kinase II. Additional studies analyzed a coding region

polymorphism in human TPH, a subtle valine to isoleucine substitution at residue

177 (V177I). Our studies suggest that this residue is involved in the optimal

x

orientation of the tryptophan substrate binding pocket in the TPH active site. These

studies have added to our understanding of the structure and function of TPH.Using

this type of information, novel TPH-specific pharmacotherapies may be developed

for use in the treatment of serotonergic disorders in human health and disease.

xi

CHAPTER I

INTRODUCTION:

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE

AROMATIC AMINO ACID HYDROXYLASES

George C.-T. Jiang

1

2

AROMATIC AMINO ACID HYDROXYLASES

Tryptophan hydroxylase (TPH; EC 1.14.16.4) is a member of a family of

enzymes known as aromatic amino acid hydroxylases (AAAH). The other members

of this family are tyrosine hydroxylase (TH; EC 1.14.16.2) and phenylalanine

hydroxylase (PAH; EC 1.14.16.1). TPH catalyzes the rate-limiting step in the

biosynthesis of the neurotransmitter serotonin (Grahame-Smith, 1964; Lovenberg et

al., 1967), and is involved in melatonin biosynthesis (Chanut et al., 2002; Privat et

al., 2002; reviewed in Martinez et al., 2001). TH is the rate-limiting enzyme in the

synthesis of the catecholamines (dopamine, epinephrine, and norepinephrine), while

PAH is rate-limiting in the synthesis of the amino acid tyrosine (for reviews, see

Hufton et al., 1995; Kumer and Vrana, 1997) and the primary enzyme responsible

for the catabolism of phenylalanine (reviewed in Martinez et al., 2001).

All three enzymes share a high degree of amino acid identity and distinct

structural and functional characteristics. Structurally, AAAH are composed of three

regions - an amino terminal regulatory domain, a carboxyl catalytic domain, and an

oligomeric binding domain at the extreme carboxyl terminus (Liu and Vrana, 1991;

Wang et al., 2000; reviewed in Ftizpatrick, 1999). While the regulatory domains

exhibit approximately 30% sequence identity, over 60% sequence identity is

exhibited in the catalytic region. The junction delineating the regulatory and catalytic

domains (Val-Pro-Trp-Phe-Pro) is completely conserved in all species of the

hydroxylases, and marks the beginning of the conserved catalytic domain (Yang et

al., 1994; Kumer et al., 1997a; Mockus et al., 1997b; Moran et al., 1998; reviewed in

3

Ftizpatrick 1999). Recent crystallographic data show that the proteins fold in a

similar manner and are virtually superimposeable in their catalytic domains. It is

generally accepted that the three hydroxylases evolved from a common progenitor

(Grenett et al., 1987; Neckameyer and White, 1992). Evidence for this is

demonstrated by the fruit fly, Drosophila melanogaster, which possesses only two

AAAH – TH and an enzyme that hydroxylates both tryptophan and phenylalanine

(Neckameyer and White, 1992).

Functionally, all AAAHs require the same co-substrates – molecular oxygen

(O2), tetrahydrobiopterin (BH4) – and a non-heme, ferrous iron co-factor to

hydroxylate their respective amino acid (Figure 1). Another similarity revolves

around the hydroxylation reaction itself, which produces water as a bi-product in all

cases. The release of water serves as the basis of one method for determining

enzyme activity. By utilizing a tritiated tryptophan substrate in the hydroxylation

reaction, one can determine enzyme activity by measuring the tritiated water

released (Beevers et al., 1983; Reinhard et al., 1986; Vrana et al., 1993). The other

method for determining enzyme activity in vitro is based on a continuous

fluorometric assay using the different spectral characteristics of tryptophan and 5-

hydroxytryptophan (Moran and Fitzpatrick, 1999). Using this assay, one directly

detects the fluorescent yield of 5-hydroxytryptophan.

4

Figure 1: Biosynthetic pathway for catecholamines and indoleamines. TH is the

rate-limiting enzyme in the synthesis of the catecholamines (DA, NE, and E), while

TPH is the rate-limiting enzyme for serotonin formation. TPH also serves as a non-

limiting enzyme in the synthesis of melatonin. Both TH and TPH require the co-

substrates molecular oxygen (O2) and tetrahydrobiopterin (BH4), as well as the co-

factor ferrous iron (Fe2+). Following hydroxylation of tryptophan at the 5' position,

aromatic amino acid decarboxylase (AAADC) decarboxylates 5-hydroxytryptophan

to form 5-hydroxytryptamine (serotonin). Serotonin is then acetylated in a rate-

limiting fashion by N-acetyltransferase (NAT) to allow for melatonin formation (not

shown). AAADC also serves to decarboxylate DOPA to form dopamine. Dopamine

is further metabolized to norepinephrine (NE) and epinephrine (E) by dopamine-β-

hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT),

respectively.

5

6

TRYPTOPHAN HYDROXYLASE AND SEROTONIN BIOSYNTHESIS

TPH is the rate-limiting enzyme in the biosynthesis of the neurotransmitter

serotonin (Grahame-Smith, 1964; Lovenberg et al., 1967). It is also involved in

melatonin biosynthesis though is not rate-limiting in that reaction. Localization of cell

bodies, as determined by in situ hydridization, illustrate that TPH mRNA-positive

neurons can be found throughout the raphe nuclei and the pineal gland (Austin and

O’Donnell, 1999). TPH is predominantly made in the central nervous system in

these brain regions (Jequier et al., 1969; Joh et al., 1975). There is also evidence

for peripheral synthesis of TPH, including cells of the enteric neurons of the gut,

platelets, retina, and thyroid cells (reviewed in Mockus and Vrana, 1998).

In mammalian species, serotonin has been detected in nearly all tissues of

the body and plays a number of different roles (reviewed in Azmitia, 2001).

Because of its important regulatory role, a number of processes have evolved to

regulate serotonin biosynthesis. In the brain, rapid regulatory control is achieved by

modulating TPH enzyme activity through phosphorylation. This mechanism of

action allows for rapid and local regulation of 5-HT biosynthesis without requiring

new enzymes to be transported from the cell body area to the synapse, which could

take hours to days to accomplish (reviewed in Azmitia, 2001).

7

SEROTONIN IN HUMAN HEALTH AND DISEASE

Serotonin (5-HT) is the neurotransmitter end-product following tryptophan

hydroxylation by TPH and subsequent decarboxylation by AAADC. Serotonin is

implicated in a variety of CNS functions such as temperature control, aggression,

pain and memory (Kandasamy and Williams, 1984; reviewed in Oliver et al., 1995;

Brentegani and Lico, 1982; and Cornwell-Jones et al., 1989). Aberrations in

serotonin synthesis, regulation, and homeostasis have been associated with

numerous psychiatric illnesses including depression, anxiety, obsessive-compulsive

disorder (OCD), bulimia, migraines and drug abuse (reviewed in Mockus and Vrana,

1998). Moreover, 5-HT itself can be oxidized in vivo by superoxide to form the toxic

metabolite, tryptamine-4,5-dione (T-4,5-D) which has been implicated in the

degeneration of serotonergic neurons following methamphetamine exposure or

cerebral ischemia reperfusion (Jiang et al., 1999).

Recent data from the National Institute of Mental Health (NIMH) demonstrate

that there is a severe socioeconomic impact of mental illness in the United States.

An estimated 22.1% of Americans suffer from a diagnosable mental disorder in a

given year (Regier et al., 1993). For example, it is estimated that 19 million adults in

the United States (10% of the US population) suffer from depression, and only one-

third of this population receives treatment. As mentioned earlier, depression is

associated with aberrant serotonergic function. Previously, treatment regimen for

depressive disorders involved tricyclic antidepressants (TCAs) which have a large

number of side effects. Today, treatment predominantly involves selective serotonin

8

reuptake inhibitors (SSRIs) and/or monoamine oxidase inhibitors (MAOIs) that

modulate neurotransmitter levels (including serotonin) with less side effects. In

2001, the pharmaceutical giants Eli Lilly and GlaxoSmithKline – producers of the

leading antidepressant drugs, Prozac (fluoxetine) and Paxil (paroxetine) – reported

$2.2 Billion and $6.2 Billion in US sales, respectively.

Typically, the treatment for serotonin-related diseases is to modulate the

serotonergic system, often by way of selective serotonin reuptake inhibitors, such as

fluoxetine or sertraline as described above. Alternative treatments, using lithium or

the former diet drug fenfluramine, work presynaptically to alter the serotonin system.

However, the side effects are vast and long-term effects remain to be determined.

TPH could contribute to the root of many of these serotonin-related disorders.

Therefore, it is necessary to further understand the structure, function and regulation

of this important enzyme to potentially provide an alternative therapeutic approach

to treat these diseases.

9

AAAH POLYMORPHISMS AND DISEASE

As the AAAHs are all rate-limiting enzymes in their respective metabolic

pathways, it is not suprising that changes in enzyme structure could change enzyme

function, and thereby manifest themselves, ultimately in abnormal clinical conditions.

This is indeed the case for PAH and TH, and subtle hints of such polymorphisms

resulting in human disease have been suggested for TPH.

It has been demonstrated that a dysfunctional or compromised PAH will result

in increased blood phenylalanine levels, or hyperphenylalaninemia (HPA) (Nowacki

et al., 1997) due to the inability to catabolize the amino acid. More severe cases of

HPA result in the condition commonly known as phenylketonuria (PKU; reviewed in

Erlandsen et al., 2001; reviewed in Jiang et al., 2001a). Researchers have identified

over 400 mutations in PAH that result in decreased enzymatic activity, and can be

linked to PKU. Unfortunately, PKU results in severe mental retardation of patients, if

left untreated. Numerous researchers have utilized PAH crystal structures

(Erlandsen et al., 1997a; Fusetti et al., 1998; Kobe et al., 1999; Jennings et al.,

2000; Miranda et al., 2002, reviewed in Erlandsen et al., 1997b) to explain the

functional effect of many of these mutations on enzymatic activity. Often, these

mutations map to important active site residues, but some map to amino acids in

structural motifs, or in important hinge structures (Bjorgo et al., 2001; Jennings et

al., 2001; Miranda et al., 2002; Horne et al., 2002).

Polymorphisms in the coding region for TH have also been identified that

result in clinical abnormalities. These point mutations in TH result in autosomal

10

recessive L-DOPA- responsive parkinsonism and dystonias (Ludecke et al., 1995,

1996, reviewed in Ichinose et al., 1999). Clinically, all of these polymorphisms

manifest themselves as motor coordination symptoms in patients as they result in

dopamine deficiency, yet without the neuronal death associated with juvenile

parkinsonism or Parkinson’s Disease. To date, only a handful of TH polymorphisms

have been described. A Glu381Lys (Q381K) change in human TH was first

described (Knappskog et al., 1995; Ludecke et al., 1995) to result in decreased TH

specific activity to about 15% of wild-type when expressed in vitro. Additionally, the

Km for the tyrosine substrate of the mutant was 6-fold higher compared to wild-type

enzyme (Knappskog et al., 1995). Similarly, a Leu205Pro (L205P) polymorphism

was described that resulted in a recombinant enzyme that exhibits 1.5% of the

activity of wild-type (Ludecke et al., 1996). Other TH polymorphisms that have been

characterized include Arg233His (R233H) (van den Heuvel et al., 1998), Cys359Phe

(C359F) (Bräutigam et al., 1999), Val112Met (V112M), and Asp498Gly (D498G)

(Wevers et al., 1999). In all cases, the effect on enzyme function can be explained

by the change in enzymatic structure, as rationalized using a crystal structure.

Due to challenges in enzyme purification and retention of activity, research on

the structure and regulation of TPH has fallen behind that of the other amino acid

hydroxylases. Only recently has the TPH crystal structure been deciphered (Wang

et al., 2002). As 5-HT plays a role in the etiology of a wide variety of neurological

diseases, but only subtle phenotypic changes occur, it has been difficult to identify

polymorphisms in the TPH gene that associate with a disease in a clear-cut manner.

Nielsen and colleagues first described an intronic polymorphism in the human TPH

11

gene (intron 7) that is associated with suicidality and variable 5-hydroxyindolacetic

acid concentrations in Finnish Caucasians (Nielsen et al., 1994). Another study has

associated another intronic TPH gene polymorphism with aggression and anger-

related traits (Manuck et al., 1999). Recently, the first coding region polymorphism

of TPH was described by Ramaekers and colleagues in a pediatric patient exhibiting

neurodevelopment problems (Ramaekers et al., 2001). Analysis of cerebral spinal

fluid (CSF) and serum from five pediatric patients exhibiting motor and

neurodevelopment problems illustrated decreased serotonin metabolites, suggesting

a deficiency in serotonin production. When treated with the end-product of TPH, 5-

hydroxytryptophan, the patients all regained normal motor and neurological function,

further suggesting a potential problem with TPH. Sequencing of the TPH gene

revealed a G529A polymorphism in the gene that would produce a subtle valine to

isoleucine substitution at residue 177 (Val177Ile) in the protein. The role of this

specific residue remains undetermined. However, a complicating factor is that the

specific Val177Ile polymorphism only occurred in one of the five patients

(Ramaekers et al., 2001).

Interestingly, however, in all the TPH-associated clinical situations, structural

and catalytic consequences of potential polymorphisms remain unknown. Through

our work, we have developed a structural model for TPH to explain the effect of

such polymorphisms, in conjunction with biochemical studies to assess the effect of

such polymorphisms on TPH function. A fundamental hypothesis is that

investigation of additional patient populations will reveal more TPH polymorphisms

that could be the cause of the neurological disorders associated with aberrant

13

PROTEIN PURIFICATION STUDIES

The first attempts to purify tryptophan hydroxylase started in the 1970s using

a number of different sources and chromatographic techniques, though there were

few publications when compared to the other AAAHs (reviewed in Cash, 1998). The

difficulty in obtaining pure TPH protein can be attributed primarily to 1) the low

quantities of TPH protein available in natural sources; and 2) the inherent sensitivity

of the enzyme to proteolytic degradation and/or inactivation (reviewed in Cash,

1998).

The initial work on tryptophan hydroxylase was focused on protein obtained

from bovine (Jequier et al., 1969; Nukiwa et al., 1974), rabbit (Tong et al., 1975;

Nakata et al., 1981; Banik et al., 1997; Moran et al., 1998), and rat (Jequier et al.,

1969; Nakata et al., 1982; Cash et al., 1985; Cash, 1998) sources. However, TPH

has been isolated from other mammalian species, including mouse (Hosada 1975;

Nakata et al., 1982b; Fujisawa et al., 1987a; Park et al., 1994), pig (Youdim et al.,

1974, 1975) and human samples (Yang et al., 1994; Kowlessur et al., 1999; Wang

et al., 2002). Successful purification of TPH was difficult as the procedures required

considerable time and extensive multi-step chromatography procedures. In fact, the

enzyme frequently lost activity and the protocols typically produced small quantities

of pure protein. Moreover, unlike more ubiquitous proteins, researchers found it

challenging to obtain significant quantities of starting material. For example, the first

purification of rat TPH used 17 rat brainstems and 9 grams of tissue yet only

produced 13.6 mg of TPH (Jequier et al., 1969). More recent purification protocols

14

entail recombinant expression of a TPH cDNA and subsequent purification, which

generates copious amounts of starting material at a very low cost. Table I also

illustrates the different yields and fold purifications that have previously been

achieved.

The recent advances in protein chemistry technologies have facilitated the

purification of greater amounts of protein, namely by the use of fusion proteins. The

first use of an affinity tag for TPH purification was by Yang and Kaufman (1994),

who produced recombinant full length and truncated human TPH fused to maltose

binding protein in E. coli. Though this technique was very useful in obtaining pure

protein, the concept of attaching a 49 kD maltose binding protein with a 2 kD linker

to TPH, or any other protein for that matter, is not optimal. Banik and colleagues

(Banik et al., 1997) utilized a much smaller fusion with an amino-terminal hexa-

histidine tag to express rabbit TPH in insect cells (Spodoptera frugiperda) for

subsequent purification. These researchers removed the hexa-histidine tag after the

purification procedures, unlike Hamdan and Ribeiro (Hamdan et al., 1999), who also

used a hexa-histidine tag (amino terminal) to express and purify E. coli expressed

rabbit TPH and TPH from the parasite Schistosoma mansoni (SmTPH). These latter

researchers were able to achieve a 45-fold purification with a yield of 0.66-0.8 mg of

purified SmTPH from 100ml of induced bacterial culture (with a specific activity of

170 nmol 5-HTP/min·mg). The recent crystallization of the catalytic core of hTPH

was also done with TPH expressed as a hexa-hisitidine fusion enzyme, although the

use of an alternative prokaryotic expression vector resulted in this enzyme being

expressed with a carboxy-terminal hexa-hisitidine tag instead of an amino-terminal

15

fusion (Wang et al., 2002). The current work has also concurrently developed a

purification system for human TPH, utilizing an amino-terminal hexa-histidine affinity

tag for rapid purification as described in Chapter 3 of this thesis.

16

17

X-RAY CRYSTALLOGRAPHIC STUDIES

Due to the inherent instability of TPH enzyme activity, large amounts of

purified protein have been difficult to obtain (Kuhn et al., 1980; D’Sa et al., 1996a,

Cash 1998). This posed a significant challenge for researchers attempting

crystallization studies. As stated above, the recent advances in protein chemistry

technologies have facilitated the purification of large amounts of protein and, thus, it

was only recently that the first crystallographic structure of TPH was determined

(Wang et al., 2002). Prior to the actual determination of the crystal structure of the

enzyme, researchers had success in determining the crystal structures for the other

members of the AAAH.

In 1997, the structure for tyrosine hydroxylase was first resolved at 2.3Å

(Goodwill et al., 1997). This structure was incomplete as it only represented the rat

catalytic domain (residues 164-498), missing the entire amino terminal regulatory

domain. This crystal structure validated the presence of a 40Å alpha helix

consisting of a 4,3-hydrophobic repeat, that was first postulated by Liu and Vrana

(1991) to be responsible for tetramerization through the interactions of anti-parallel

coiled coils. The crystal structure also revealed potential intersubunit binding

domains, such as a dimerization interface between two TH monomers formed by a

salt bridge between Lys170 and Glu282 and adjacent hydrogen bonding

interactions. This interaction is conserved in TPH and was studied by Yohrling and

colleagues (Yohrling et al., 1999; Yohrling et al., 2000). Shortly following the report

of the initial TH crystal structure, the second crystal structure of TH was resolved to

18

2.3Å by Goodwill and colleagues (1998). This new structure was also truncated,

missing the entire regulatory domain (N∆1-159). Nonetheless, the structure did

provide new insights to the enzyme, as it was resolved with iron and a pterin

cosubstrate analogue in the active site (Goodwill et al., 1998).

The number of published crystal structures for phenylalanine hydroxylase is

significantly greater. The first PAH crystal structure was determined at about the

same time as that for tyrosine hydroxylase in 1997. This crystal structure was a

homodimer but truncated, only representing the catalytic domain of human PAH

(residues 117-424, Erlandsen et al., 1997a). Soon after, the structure of the

catalytic domain of human PAH (residues 118-452) in its tetrameric form was

resolved (Fusetti et al., 1998). Additionally, researchers were able to determine the

structure of the human PAH catalytic domain to 2.0Å (residues 117-424) with

various catechol inhibitors bound (Erlandsen et al., 1998). These structures gave

insight into residues in the active site that might play a role in substrate binding and

catalysis. A more complete structure for PAH was resolved to 2.2Å with the

crystallization of rat PAH (residues 1-429) that was missing only the carboxyl

terminal tetramerization domain (Kobe et al., 1999). Interestingly, it was observed

that phosphorylation did not result in dramatic structural changes, suggesting subtle

allosteric regulatory mechanisms for PAH following phosphorylation.

The structure for pterin-bound human PAH (residues 118-424) was

determined by Erlandsen and colleagues in 2000 (Erlandsen et al., 2000). This

provided a more detailed view into the active site of the enzyme, and the potential

mechanism of catalysis. Soon thereafter, the structure of the human PAH catalytic

19

domain (N∆1-102/C∆428-452) with catalytically active Fe(II) and in a binary complex

with the natural cosubstrate, BH4, was resolved (Andersen et al., 2001). This has

been followed up with a report of the structure of a truncated human PAH enzyme

(N∆1-102/C∆428-452) ternary complex with BH4 and 3-(2-thienyl)-L-phenylalanine

(Andersen et al., 2002). Further insights into understanding PAH, and by extension,

understanding the structure and function of TH and TPH, were provided by the

comparison of bacterial versus human PAH (Erlandsen et al., 2002). These

researchers observed similar folding in the catalytic domain, but different enzyme

stability and reaction rates between the prokaryotic and eukaryotic enzymes.

Recently, researchers have investigated the role of the amino terminus of

PAH, which has provided no interpretable electron density in previous PAH

crystallographic studies. This N-terminal sequence had been surmised to play a role

in autoregulation of PAH, based on the crystal structure of PAH (Kobe et al., 1999)

and deletion mutagenesis (Jennings et al., 2001, and Wang et al., 2001; Horne at

al., 2002). Using NMR, Horne and colleagues demonstrated that the N-terminus is

a mobile flexible region that associates with the folded core of the molecule after

binding of the phenylalanine substrate, the association of which can be facilitated by

phosphorylation (Horne at al., 2002).

In spite of intensive efforts by several groups, no successful crystallization of TPH

occurred until recently (Wang et al., 2002). In advance of such a structure, and

given the high sequence homology between the AAAHs, the present work includes

the construction of the first hypothetical molecular model of human TPH (Vrana,

1999; Jiang et al., 2000b; Figure 2) based upon the then available crystal structures

20

of both rat TH and rat PAH (Goodwill et al., 1997; Kobe et al., 1999). Due to the

challenges involved in getting crystal formation with all domains present, the

hypothetical model was, and still remains, the most comprehensive structure of any

AAAH to date as it is nearly full length (missing only one amino acid residue). This

model also has significant advantages over an automated model of TPH generated

by SWISS-MODEL (Guex et al., 1997, 1999). This latter structure was truncated

(containing residues 104-411), and not subject to rigorous investigator-interactive

energy minimizations used in the present work. Potential functional motifs, as well

as amino acids involved in substrate binding and phosphorylation, have been

identified with the use of our hypothetical model. The recent crystallization of a

truncated form of human TPH (N∆1-102/C∆402-444) at a resolution of 1.7Å and

with bound cofactor analogue (7,8-dihydro-L-biopterin), validates the catalytic

domain of this hypothetical model and provides new insights into tryptophan

hydroxylation. An “iterative magic fit” of the TPH crystal structure and hypothetical

model using Swiss-PdbViewer (Guex and Peitsch, 1997) indicate a root mean

square deviation (R.M.S.D.) of 1.02 Å between the carbon backbones of the

structures.

21

Figure 2: Structural Comparisons of the AAAHs. The crystal structures of PAH and

TH are shown beside a predicted structure of the TPH catalytic domain. These

structures only depict the catalytic domains of these AAAHs. The PAH structure

(PDB entry 1PAH) depicts residues 117 to 424, thereby missing the entire regulatory

domain and the extreme carboxy terminal. The TH structure (1TOH) depicts

residues 164 to 498, and is also missing the entire regulatory domain. The

predicted TPH catalytic domain was generated by homology-based modeling in the

“Composer” module of Sybyl 6.5 (Tripos, St. Louis, MO). This figure was originally

generated by Mr. Jiang and is reproduced from Vrana, 1999.

22

23

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33

STATEMENT OF PURPOSE

The goal of this dissertation research was to utilize recombinant DNA

technologies as well as homology-based computer modeling to more fully

understand the structure and function of human TPH. The studies presented in the

next chapters are intended to advance our characterization of TPH, and the subtle

role that mutations in the enzyme may play in disease.

The first objective was to generate a hypothetical three-dimensional model of

human TPH using homology-based molecular modeling in the absence of an

experimentally-determined crystal structure. Previous in vitro structural data of TPH

and the other AAAHs were mapped to this model. Our hypothetical model revealed

information regarding the differential regulation and function of TPH when compared

to previously determined structures for TH and PAH. Thereafter, we performed site-

directed mutagenesis to identify previously unidentified functional domains within the

TPH protein, and to validate the hypothetical model.

The second objective was to perform in vitro expression analysis of a

naturally-occuring mutation in human TPH. The rationale for doing so is the same as

those behind the analysis of PAH polymorphisms related to the metabolic condition

PKU. That is, to confirm that a “disease-associated” mutation is truly pathogenic, to

assess the severity of a mutation’s impact, and to elucidate the molecular

mechanisms involved by understanding how a mutation exerts its deleterious

effects. In the course of these experiments, we developed a bacterial expression

system that permits the rapid purification of the human TPH enyme.

34

Overall, these studies have revealed that the structure of TPH closely

resembles other members of the AAAH superfamily, and have illuminated new

insights into the structural and functional aspects of the protein. Additionally, these

studies indicate that the first described coding region polymorphism of TPH may

have a minimal impact on enzyme activity. However, it has identified what is

important new information on structural determinants of the TPH active site.

CHAPTER II

IDENTIFICATION OF SUBSTRATE ORIENTING AND PHOSPHORYLATION

SITES WITHIN TRYPTOPHAN HYDROXYLASE USING HOMOLOGY-BASED

MOLECULAR MODELING

George C.-T. Jiang, George J. Yohrling IV, Jeffrey D. Schmitt,

and Kent E. Vrana

The following chapter is a paper published in Journal of Molecular Biology, volume

302, pages 1005-1017 (2000). The materials have been reprinted with permission.

Stylistic variations are due to the requirements of the journal. The original proposal

and concept for these studies were those of George Jiang, George Yohrling, and

Kent Vrana. George Jiang and George Yohrling contributed equally to construction

of the hypothetical model and preparation of the manuscript. Dr. Kent E. Vrana

served in an advisory position on experimental design and manuscript preparation.

Dr. Jeffrey D. Schmitt (Targacept Inc.) provided technical assistance and scientific

advice with the molecular modeling software and aided with construction of the

hypothetical model. All final energy minimizations of the model were performed by

Dr. Schmitt.

35

36

Abstract

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the

biosynthesis of serotonin (5-HT). The inherent instability of TPH has prevented a

crystallographic structure from being resolved. For this reason, multiple sequence

alignment-based molecular modeling was utilized to generate a full-length model of

human TPH. Previously determined crystal coordinates of two highly homologous

proteins, phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), were

used as templates. Analysis of the model aided rational mutagenesis studies to

further dissect the regulation and catalysis of TPH. Using rational site-directed

mutagenesis, it was determined that Tyr235 (Y235), within the active site of TPH,

appears to be involved as a tryptophan substrate orienting residue. The mutants

Y235A and Y235L displayed reduced specific activity compared to wild-type TPH

(≈5% residual activity). The Km of tryptophan for the Y235A (564 µM) and Y235L

(96 µM) mutant was significantly increased compared to wild-type TPH (42 µM). In

addition, kinetic analyses were performed on wild-type TPH and a deletion construct

that lacks the amino terminal autoregulatory sequence (TPH N ∆15). This sequence

in phenylalanine hydroxylase (residues 19-33) has previously been proposed to act

as a steric regulator of substrate accessibility to the active site. Changes in the

steady-state kinetics for tetrahydrobiopterin (BH4) and tryptophan for TPH N ∆15

were not observed. Finally, it was demonstrated that both Ser58 and Ser260 are

substrates for Ca2+/calmodulin-dependent protein kinase II. Additional analysis of

this model will aid in deciphering the regulation and substrate specificity of TPH, as

37

well as providing a basis to understand as yet to be identified polymorphisms.

38

Introduction

Tryptophan hydroxylase (TPH; EC 1.14.16.4) is a member of the aromatic

amino acid hydroxylase superfamily (Fitzpatrick, 1999). The other members of this

family include tyrosine hydroxylase (TH; EC 1.14.16.2) and phenylalanine

hydroxylase (PAH; EC 1.14.16.1). The enzymes share approximately 50% amino

acid sequence identity and are thought to have evolved from a common progenitor

(Grenett et al., 1987; Neckameyer & White, 1992). TPH serves as the rate-limiting

enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT), and as a non-

limiting component of the melatonin synthetic pathway within the pineal gland.

Recent evidence has shown TPH to be rate-limiting for melatonin biosynthesis in the

retina (Thomas et al., 1998). Within the central nervous system, TPH is localized in

the raphe nuclei, as well as in various peripheral locations such as the pineal gland,

enteric neurons of the gut, and the enterochromaffin mast cells of the intestine and

pancreas (Mockus & Vrana, 1998). Serotonin is an important monoamine that has

been implicated in a wide variety of central nervous system functions including

temperature control, aggression, pain, and memory (Kandasamy & Williams, 1984;

Brentegani & Lico, 1982; Cornwell-Jones et al., 1989; Olivier at al., 1995).

Aberrations in 5-HT synthesis and regulation have been linked to a diverse group of

neurological and psychiatric disorders such as Parkinson’s disease, Gilles de la

Tourette’s syndrome, multiple sclerosis, depression, and obsessive compulsive

disorder (Volicier et al., 1985; Schauenburg & Dressler; 1992; Tabaddor et al., 1978;

Mann et al., 1997; Delgado & Moreno, 1998). TPH could be a potential

39

pharmacotherapeutic target in the etiology of several of these neurological diseases.

The hydroxylases are composed of an amino terminal regulatory domain and

a carboxyl terminal catalytic domain (Grenett et al., 1987; Ledley et al., 1985;

Darmon et al., 1988; Daubner et al., 1993). It is presumed that the regulatory

domains of the enzymes act to modulate activity. Ser58, the one documented

phosphorylation site in TPH resides in this regulatory domain (Kuhn et al., 1997;

Kumer et al., 1997). The active sites reside within the catalytic domain and are

responsible for the hydroxylation of their respective aromatic amino acid residues.

Chimeric hydroxylases have been created to demonstrate that substrate specificity

is controlled by the catalytic domain (Mockus et al., 1997b; Daubner et al., 1997).

Table 1 summarizes all of the published recombinant TPH mutants that have been

created to dissect the structure, function, and regulatory mechanisms of TPH.

TPH is known to exist both in vivo and in vitro as a homotetramer (reviewed in

Hufton et al., 1995). Recently, this laboratory has identified intersubunit binding

domains in both the regulatory and catalytic domains of the rabbit TPH protein

(Mockus et al., 1997a; Yohrling et al., 1999). These interactions are thought to be

hydrophobic and necessary for the proper macromolecular assembly of TPH.

Despite the high level of protein sequence identity in the catalytic domains of TH and

TPH, they do not form heterotetramers (Mockus et al., 1998). It is hypothesized that

there are additional structural domains within TPH that work in tandem with those

already identified, to allow for its regulation. This is an additional reason that

crystallization or theoretical modeling of TPH is of particular significance.

Knowledge (or prediction) of a detailed quaternary structure will enable design of

40

potential pharmacotherapeutics to modulate TPH activity. Moreover, a detailed

tertiary and quaternary structure of TPH will provide insight into the factors

governing differential substrate specificity among the aromatic amino acid

hydroxylases.

41

42

It has long been known that point mutations within the PAH gene lead to

insufficient PAH activity and the build-up of the amino acid phenylalanine. This

leads to the metabolic imbalances and mental retardation that are characteristic of

phenylketonuria (PKU). There are in excess of 300 known distinct mutations in PAH

that lead to PKU (Eisensmith & Woo, 1991). Recently, point mutations in the

catalytic domain of TH have been linked to Segawa’s syndrome and L-dopa

responsive Parkinsonism (Ludecke et al., 1995, 1996; Knappskog et al., 1995). In

addition, an intronic polymorphism in the human TPH gene has been discovered

that is associated with suicidality and variable 5-hydroxyindolacetic acid (5-HIAA)

concentrations in Finnish Caucasians (Nielsen et al., 1992, 1994). To date, it is

unclear what, if any, such an intronic polymorphism would have on TPH regulation.

However, the genetic findings in PAH and TH suggest that point mutations in critical

regulatory and structural domains of TPH will be discovered and that these

mutations may explain selected neurological disorders associated with aberrant

serotonergic levels.

It is unfortunate that research advances on TPH have lagged behind that of

the other aromatic amino acid hydroxylases. The extreme instability of TPH has

precluded its purification in large quantities (Kuhn et al., 1980; D’Sa et al., 1996a,b;

Cash, 1998; Moran et al., 1998), and without adequate purification, a complete

crystallographic model for TPH will not be elucidated. For this reason, it has

become important to apply the existing information on the related aromatic amino

acid hydroxylases (PAH and TH) to aid in characterizing the structure and regulation

of TPH. The recent crystallization of both PAH and TH provide much of this

43

information (PAH; Erlandsen et al., 1997a,b, 1998, 2000; TH; Goodwill et al., 1997,

1999).

Here, we present a detailed structural model of human TPH, based on the

existing structural coordinates for TH and PAH (1TOH and both 1PHZ and 1PAH,

respectively; Research Collaboratory for Structural Bioinfomatics Protein Databank;

RCSB-PDB) generated using multiple sequence alignment homology modeling

techniques (Blundell et al., 1988; Sutcliffe et al., 1987). In addition to being stable

relative to molecular mechanics, our model was utilized to successfully predict

aspects of TPH assembly and function.

44

Results

General topography of the hypothetical TPH model

The fully minimized, hypothetical human TPH model is shown in Figure 1.

The monomeric unit contains both the amino terminal regulatory domain and the

carboxy terminal catalytic domain. The amino terminal end of the protein lacks an

organized secondary structure. Previously determined structural and regulatory

moieties are evident on the model. A Ramachandran plot (PROCHECK analysis at

2.0 Å) indicates that 84.7, 14.0, 1.0, and 0.3% of non-glycine and non-proline

residues reside in most favorable, additionally allowed, generously allowed and

disallowed regions respectively. Notably, the last category (disallowed) is composed

of a single amino acid (Ile 2) at the disordered amino terminus. In Figure 2, the high

degree of secondary and tertiary structural identity between TPH, PAH and TH is

evident in a side-by-side comparison of all three structures. Slight structural

variations are present between the regulatory domains of PAH and TPH. Figure 3

depicts a theoretical tetramer of human TPH. The tetramer is anchored by the C-

terminal tetramerization domain that is composed of hydrophobic, coiled-coil

interactions. The regulatory domain of a given monomer forms intersubunit contacts

with both the tetramerization domain and regulatory domain of an adjacent subunit.

45

Figure 1: Hypothetical model of human TPH (monomer). A 3-D model of the

monomeric subunit of human TPH is depicted. All amino acids were modeled

except for the initial methionine residue. Secondary structure is identified by color:

β-sheets (yellow), α-helix (pink), and random coil (white). The locations of Ser58,

Ser260, and Tyr235 are indicated. The amino terminus of TPH begins with a non-

structured random coil formation termed the active-site gate. The carboxy terminal

helix (tetramerization domain) and the amino terminal 4,3-repeat have previously

been identified as structural motifs required for the proper macromolecular assembly

of TPH. A proposed interface domain between the regulatory and catalytic domain

of TPH is also indicated. The model was generated on a Silicon Graphics

Workstation with the modeling program Sybyl 6.5 (Tripos Inc., St. Louis, MO).

46

47

Figure 2: Side by side comparison of TH, PAH, and hypothetical TPH. The

previously resolved crystal structures for rat PAH (1PHZ) and rat TH (1TOH) are

shown along with the full length human TPH model. The X-ray diffraction

coordinates for both 1PHZ and 1TOH were used to perform homology based

modeling. A high degree of secondary and tertiary structure identity among all the

hydroxylase models is evident.

48

49

Figure 3: Tetramer of hypothetical TPH. Each monomer of TPH is shown in a

different color. The tetramer is anchored by a carboxy terminal leucine zipper and

additional intersubunit hydrophobic interactions between the regulatory domains.

The model was created using an alignment based on the previously reported

tetramer for TH (Goodwill et al., 1997).

50

51

TABLE 2. Steady-state kinetic parameters of wild-type TPH and mutants

Enzyme BH4 Km (µM) L-tryptophan Km (µM)

wt TPH 61 ± 19 30 ± 16

N ∆ 15 50 ± 6 41 ± 13

C ∆ 17 55 ± 4 30 ± 6

wt TPH 78 ± 0.4 42 ± 2

Y235A 34 ± 11 564 ± 85*

Y235L 56 ± 9 96 ± 5*

TPH activity was determined (see Materials and Methods) and the kinetic constants were

calculated by non-linear regression analysis. The substrate concentrations were 100 µM

BH4 (tryptophan variable) and 50 µM tryptophan (BH4 variable). The mean values for BH4

and tryptophan were compared independently of each other. * One-way ANOVA, followed

by a Student’s Newman-Keuls test at 95% confidence indicated that the kinetic differences

observed were statistically significant (p < 0.05).

52

Figure 4: Phosphorylation and immunodetection of TPH. (a) SDS-PAGE analysis of

CaMPKII phosphorylated TPH proteins. Lane1: pET-21c negative control; lane 2:

wild-type TPH; lane 3: S58A; lane 4: S260A; lane 5: S58A/S260A. A decrease in

the amount of incorporated phosphate is observed in the double point mutant

S58A/S260A. (b) Immunoblot of phosphorylated TPH. Immunoreactive proteins

were detected with a monoclonal antibody (WH3) at a 1:1000 dilution followed by an

anti-mouse IgG-HRP from sheep at a 1:1500 dilution. Each protein migrated with a

molecular weight of 51 kDa. No immunoreactive protein was detected in the pET -

21c control.

53

54

Kinetic analysis of autoregulatory sequence

Amino terminal deletion of the proposed active-site guard (residues 1-15;

N∆15) and subsequent kinetic analysis produced no change in the Km of TPH for

tryptophan (41(±13) µM) when compared to wild-type TPH (30(±16) µM; Table 2).

Likewise, no alteration in the Km for BH4 was observed in N ∆15 when compared to

TPH. A similar analysis was performed on a TPH mutant that lacked the C-terminal

tetramerization domain. Non-significant changes in the Km for both BH4 and

tryptophan were observed for the truncated mutant enzyme (see Table 2).

Phosphorylation of TPH by CaMPKII

The hypothetical model indicates that Ser260 resides on the exterior of the

TPH monomer. It has been postulated that Ser260 may serve as a substrate for

phosphorylation by CaMPKII (56) because it is located within the consensus motif

for this kinase (Pearson & Kemp, 1991). To determine whether Ser260 is

phosphorylated by CaMPKII, a series of point mutations were created to identify the

phosphoryl accepting residue. In Figure 4(a), a phosphorylated band at 51 kDa,

corresponding to the molecular weight of TPH is observed for wild-type TPH, S58A,

and S260A. However, little phosphorylation is apparent in the mutant S58A/S260A.

An unknown bacterial protein (≈49 kDa) was phosphorylated (Figure 4(a)), but

lacked immunoreactivity (Figure 4(b)). The data indicate that both Ser58 and Ser260

are substrates for CaMPKII. The corresponding western blot for the phosphorylation

reaction is depicted in Figure 4(b) and demonstrates that comparable amounts of

55

TPH protein are present in all samples (even the unlabeled S58A/S260A double

point mutation), and all TPH proteins migrated with a molecular weight of 51 kDa.

Rational site-directed mutagenesis

Analysis of the TPH model and a PAH structure with bound inhibitor (6PAH;

Erlandsen et al., 1998) identified Tyr235 (Y235) in TPH as a potential tryptophan

substrate orienting residue. It was determined that the side-chain of the

corresponding active-site residue in 6PAH (L248) is 5.6 Å from the bound catechol

inhibitor (Erlandsen et al., 1998). Site-directed mutagenesis of Y235 to an alanine

residue (Y235A) and Y235 to a leucine residue (Y235L) significantly reduced TPH

enzymatic activity when compared to wild-type TPH at near saturating substrate

concentrations (p < 0.0005). Figure 5 shows the specific activities for wild-type

TPH, Y235L, and Y235A. The values have been normalized for the amount of

immunoreactive protein present via densitometric analysis (hence enzyme activity

units of nmol hr-1 ROD-1). Steady-state kinetic analyses were then performed to

investigate the nature of the reduction in activities for the Y235 mutations. The Km

of Y235A (564(± 85) µM) and Y235L (96(± 5) µM) for tryptophan were both

dramatically increased over wild-type TPH (42(± 2) µM). Moreover, the Km of the

mutants, for BH4, were unchanged (wild-type TPH, 78(± 0.4) µM; Y235A, 34(± 11)

µM; Y235L, 56(± 9) µM) (see Table 2). Tryptophan substrate inhibition was not

observed in Y235A (Figure 6) or Y235L (data not shown).

56

Figure 5: Specific activities of TPH active-site mutations. Mutation of tyrosine 235

to an alanine residue (Y235A) and a leucine residue (Y235L) reduced the specific

activity (nmol hr-1 ROD-1) of TPH by 94 and 95%, respectively. To determine these

values, constant amounts of the three recombinant enzymes (as determined via a

radioenzymatic activity assay) were applied to a SDS-PAGE gel and electroblotted.

The blot was probed with a monoclonal anti-TPH antibody (WH3; Sigma) followed

by a sheep anti-mouse IgG HRP-coupled secondary antibody (Amersham). The

activity was then normalized for the amount of immunoreactive protein present in

each sample by densitometric analyses (O’Neill et al., 1989). This method accounts

for differences in bacterial expression and/or liberation of the recombinant proteins

such that enzymatic activities can be directly compared. Shown here is the specific

activity mean ± S.E.M. from three independent experiments.

57

58

Figure 6: Lineweaver-Burk analysis of wild-type TPH and TPH Y235A. Shown is a

representative Lineweaver-Burk plot for wild-type TPH (solid circles ) and TPH

Y235A (solid triangles ▼). Tryptophan concentrations ranged from 10 µM to 1mM.

For wild-type TPH, regression analysis was performed after omitting the activity

values recorded at 400 µM and 1mM due to significant TPH inhibition at these

concentrations. For Y235A (this plot) and Y235L (data not shown), no inhibition of

TPH activity was recorded at tryptophan concentrations of 400 µM or 1mM. In this

particular experiment, TPH had a Km for tryptophan of 44 µM, while Y235A was 730

µM.

59

60

Discussion

Our understanding of the aromatic amino acid hydroxylases has increased

greatly in recent years. This progress culminated in the crystallization of the

catalytic core of TH (Goodwil et al., 1997) and the first crystallization of a PAH

regulatory domain (Kobe et al., 1999). Due to the extreme instability of TPH and the

resulting inability to obtain significant amounts of purified protein, a crystal structure

for TPH remains elusive. TPH shares a high degree of sequence identity with both

TH and PAH (Grenett et al., 1987; Neckameyer & White, 1992). For this reason, we

have utilized the crystal coordinates for both TH (1TOH) and PAH (1PHZ and 1PAH)

to perform multiple sequence alignment-based molecular modeling of the human

and rabbit TPH proteins. A Blast comparison (Altschul et al., 1990) of the rabbit and

human protein sequences, yields 97% overall sequence identity. Only 11 out of the

444 amino acid residues are different and only four of these amino acid residues are

non-conservative substitutions. With such a high degree of sequence identity, and

no mutations in the active-site region which could cause putative changes in

structure/function, only the human TPH model was subjected to further energy

minimizations to make predictions with respect to aspects of catalysis and

regulation.

It is noteworthy to mention that Swiss-Model, the automated protein modeling

server, (GlaxoWellcome Experimental Research Center in Geneva, Switzerland) has

previously generated a model of TPH (Guex & Peitsch, 1997; Peitsch, 1995, 1996).

This model is based on several phenylalanine hydroxylase structures (1PHZ, 2PHM,

61

1PAH, 3PAH, 4PAH), but is truncated at the carboxy terminus. Furthermore, it was

generated without human intervention and interpretation. PROCHECK analysis

indicates similar Ramachandran properties as our manually predicted human TPH

model (85.1% most favored, 11.7% additionally allowed, 2.4% generously allowed,

and 0.8% in disallowed regions) but with a greater number of residues in generously

allowed and disallowed regions. In addition, the Swiss-Model structure has 46 bad

contacts, a significantly greater number than the 2 that our model exhibits.

The human 3-D TPH model presented here is the first full-length TPH

structure to be reported. As expected from the modeling approach, the human TPH

monomer has significant structural identity to the previously crystallized proteins, TH

and PAH (Figure 2). The monomer of TPH has an amino terminal regulatory

domain (residues 2-106), a carboxy terminal catalytic domain (residues 107-423),

and a tetramerization domain (424-444). This regulatory and catalytic domain

organization is consistent with experimental observations of the independent

catalytic activity of the C-terminal portion of the enzyme (Kumer et al., 1997; D’Sa et

al., 1996a; Yang & Kaufman, 1994). The tetramerization domain of TPH is

composed of an extreme C-terminal 4,3-hydrophobic repeat. Interspersed within

this repeat is a leucine zipper that was originally postulated by Liu and Vrana (1991)

to be important for the tetramerization of the AAAH. Deletion mutagenesis of this

domain resulted in dimers and monomers of TPH that retained activity (Mockus et

al., 1997a; D’Sa et al., 1996b). Kinetic analysis of the mutant C ∆17 TPH, that lacks

this tetramerization domain, failed to show a significant change in the Km of C ∆17

TPH for BH4 or tryptophan (Table 2). These data suggest that the tetramerization

62

domain of TPH is a structural motif that is not required for activity. The role of

oligomerization in regulatory mechanisms of TPH and the other hydroxylases remain

to be determined.

Our human TPH model predicts an alternative regulatory domain orientation,

relative to PAH and the Swiss-model predicted TPH structure. According to the

model, the entire regulatory domain is rotated away from the catalytic domain. In

PAH (1PHZ), three regions of interfacial contact are observed (46-65, 107-117, 412-

414 possessing 55%, 75%, and 67% sequence identity to TPH, respectively). Our

findings suggest that the numerous interfacial domain contacts are more solvent

exposed in TPH. The absence of these interface interactions in TPH may be a

result of the multiple sequence alignment-based modeling. It was least successful in

generating the former two regions where loop searching was required to fill the gaps

(Figure 7). If this alternative N-terminal domain orientation proves to be correct, it

could have implications for protein stability (Mockus et al., 1997b; Daubner et al.,

1997) and/or regulation.

Kobe et al. (1997a) have hypothesized that an amino terminal autoregulatory

sequence is present in PAH. These amino acids in PAH (19 to 33) work to limit

substrate accessibility into the active site. This sequence is homologous to amino

acids 1 to 15 in TPH. Upon the deletion of the proposed active-site gate in TPH (as

with the mutant N ∆15), the entrance proposed by Kobe and colleagues is no longer

sterically inhibited. This should permit free substrate access and increase the

chances of the slow step of BH4 binding to occur as previously described for TH

(Fitzpatrick, 1988). However, such a mutation did not alter the steady-state kinetics

63

of TPH. Therefore, it is unlikely that an autoregulatory sequence, as observed in

PAH functions in TPH. Instead, it appears as though access to the active site can

be accomplished from the opposite side of the catalytic domain in addition to the

previously described 12 Å active-site entrance for TH (Goodwill et al., 1997). This

ready access may explain why no alteration in Michaelis-Menten constants was

observed. However, more rigorous analyses are needed to verify this hypothesis.

Similar studies in PAH must be performed to determine the exact function of its

amino terminal autoregulatory sequence.

Recent studies of the AAAH have identified several structural motifs important

in macromolecular assembly. It has been proposed that a 4,3-hydrophobic repeat in

the regulatory domain of TPH (residues 21-41) exists as an alpha helix to permit

intersubunit hydrophobic interactions that support assembly (Yohrling et al., 1999).

The present model verifies this earlier prediction that a portion of these amino acid

residues exist as an alpha helix on the exterior of the subunit (Figure 1). In a similar

way, the tetramerization domain (leucine zipper) that was hypothesized by Liu and

Vrana (1991) to exist has been demonstrated for both TH (Goodwill et al., 1997) and

PAH (Fusetti et al., 1998). The model also predicts that a parallel alpha helix within

the regulatory domain (amino acid residues 76-85) could function as an intersubunit

binding domain to allow for the tetramerization of TPH. A Blast comparison (Altschul

et al., 1990) of the TH and TPH protein sequences yields only 30% overall sequence

identity in this region. The structure of the homologous region in TH remains to be

determined. However, such differences in amino acid identity could account for the

64

homotetramerization of TH and TPH, as could the different splice variants of human

tyrosine hydroxylase.

Numerous studies have shown TPH to be phosphorylated and/or activated by

CaMPKII (Kuhn et al., 1980; Furukawa et al., 1993; Ichimura et al., 1987; Ehret et

al., 1989; Yamauchi et al., 1981). However, TPH activation requires the presence of

the activator protein 14-3-3 (Ichimura et al., 1987; Yamauchi et al., 1981). So far,

no studies have identified the specific CaMPKII substrate residue(s) within TPH.

The human TPH model and the initial rabbit TPH structure (data not shown) suggest

that Ser260 could be a substrate for CaMPKII, due to its external orientation on the

TPH monomer (Figure 1). The phosphorylation studies presented here verify these

predictions by demonstrating that Ser58 and Ser260 are phosphorylated by

CaMPKII. The double point mutant S58A/S260A appears to incorporate a minimal

amount of phosphate. This may result from the partial phosphorylation of Ser443 in

denatured TPH. Ser443 also resides in the midst of a consensus sequence for

CaMPKII (Hufton et al., 1995). However, Ser443 is engaged in the strong

hydrophobic interactions of the tetramerization domain. It is therefore unlikely that

CaMPKII has ready access to this residue under non-denaturing conditions. The in

vitro activation of recombinantly expressed TPH and its mutants, in the presence of

both CaMPKII and 14-3-3 remains inconclusive (data not shown).

The human TPH model presented here was successful in predicting an

aspect of TPH active-site structure and specificity. Based on previous findings in TH

and PAH, as well as primary sequence alignments, the predicted TPH active-site

topology suggested that Y235 (corresponding to L294 in TH and L248 in PAH) could

65

serve to interact with and orient the tryptophan substrate. Mutagenesis of Y235

resulted in a significant reduction of TPH activity (≈95%). Kinetic analyses

confirmed our hypothesis that Y235 is a tryptophan orienting residue. Both mutants

(Y235A and Y235L) exhibit a dramatic increase in the Michaelis constant for

tryptophan, but not BH4. Mutagenesis of this residue also eliminates the substrate

inhibition of TPH observed at high levels (>200 µM) of tryptophan (Figure 6). The

corresponding identity of Y235 in both TH and PAH is leucine. TPH Y235 is located

in a region of 21 amino acid residues, at the entrance of the active site (active site

loop and α6), that exhibits an extraordinary amount of amino acid identity (86%) for

all aromatic amino acid hydroxylases (Figure 8). It is hypothesized that the longer

side chain of leucine is required to hold the smaller amino acid substrates,

phenylalanine and tyrosine, in the optimal orientation within the active sites of PAH

and TH respectively. The aromatic ring of Tyr235 in TPH may form a π-stacking

interaction with tryptophan. This type of interaction appears to play a significant role

in mediating substrate specificity of TPH.

In addition to the data presented here, the hypothetical model of human TPH

will serve numerous other capacities. First, the molecular model will aid in the

development of novel pharmacotherapeutics. The availability of a detailed active

site conformation could help identify a new generation of highly specific inhibitors of

TPH for the potential treatment of CNS disorders associated with excessive

serotonergic activity (e.g., restless leg syndrome). Secondly, detailed modeling of

TPH will assist biogenic amine research by identifying a list of potentially interesting

residues that may be involved in the regulation and activity of TPH, thus assisting

66

rational site-directed mutagenesis studies. Third, the new model may function as

the template on which as yet to be identified polymorphisms can be explained.

Single nucleotide polymorphisms (SNPs) are known to play a pivotal role in human

health and disease (Tomita-Mitchell et al., 1998; Uglialoro et al., 1998). This is

particularly true of PAH where 330 independent polymorphisms have been reported

that alter enzyme activity (Kobe et al., 1999). The present model provides a

platform on which to base structural predictors for TPH following identification of the

mutations. It is hoped that one day, clinical screening will be available to identify

potentially deleterious polymorphisms in TPH to aid in the prevention and treatment

of the numerous neurological diseases associated with aberrant 5-HT and TPH.

67

Figure 7: Multiple sequence alignment. A multiple sequence alignment of the

template crystal structures 1PHZ, 1PAH and 1TOH and human TPH (1TPH). This

alignment was generated by the Composer module of Sybyl 6.5 (Tripos Inc., St.

Louis, MO) and used to construct the hypothetical human TPH model. Residues in

red indicate where a gap resulted in the preliminary model, which were subsequently

completed by the loop search function. Bolded residues indicate amino acids within

the interface between the regulatory and catalytic domains as observed in PAH

(1PHZ). Residue numbering is according to the human TPH amino acid sequence.

68

69

Figure 8: Sequence alignment of the aromatic amino acid hydroxylases. Amino

acid alignments for human TPH (Accession number; P17752), rabbit TPH (P17290),

rat TH (P04177), human TH (P07101), rat PAH (P04176), and human PAH

(P00439) for a 21 amino acid stretch that spans the beginning of the active sites for

each of the enzymes. 86% amino acid identity is observed in this region. The

overall identity of the catalytic domains of the hydroxylases is significantly lower

(≈50%). Tyrosine 235 (Y235) of rabbit TPH was mutated to both an alanine and a

leucine residue. Above the amino acid residues, lines indicate the regions

corresponding to the active-site loop and alpha helix 6 (α6). The active sites of the

hydroxylases are thought to contain helices α6-α9 (Erlandsen et al., 1997a).

70

71

Materials and Methods

Materials

All materials were obtained from the Sigma Chemical Company (St. Louis,

MO) with the following exceptions: restriction and DNA modifying enzymes were

supplied by Promega Corporation (Madison, WI) and New England Biolabs (NEB;

Beverly, MA); L-[5-3H] tryptophan and [γ-32P] ATP from Du Pont NEN Research

Products (Boston, MA); IPTG, ampicillin, and activated charcoal (Darco G-60) from

Fisher Scientific (Pittsburgh, PA). The pET-21c vector and BL21[DE3](F- OmpTrB-

mB-) competent cells were acquired from Novagen Corporation (Madison, WI).

Reinforced nitrocellulose (Duralose-UVTM) and the QuikChangeTM site-directed

mutagenesis kit was purchased from Stratagene (La Jolla, CA). The Benchmark

prestained protein ladder was purchased from Life Technologies (Gaithersburg,

MD). Chemiluminescent reagents (RenaissanceTM) were purchased from NEN

Research Products (Boston, MA). The mouse anti-TPH monoclonal antibody (WH3)

was obtained from Sigma-RBI (St.Louis, MO). The sheep anti-mouse IgG HRP-

coupled secondary antibody was purchased from Amersham Life Sciences

(Arlington Heights, Il). Ca2+/Calmodulin-dependent protein kinase II α-subunit was

purchased from Calbiochem (San Diego, CA). Finally, all sequencing was

performed by the DNA Sequencing and Gene Analysis Facility of the Molecular

Genetics Program (Wake Forest University School of Medicine) using a Perkin

Elmer/Applied Biosystems 377 Prism automated DNA sequencer.

72

Molecular modeling

The three-dimensional models of human and rabbit tryptophan hydroxylase

(accession numbers NP_004170 and P17290 respectively) were generated using

the multiple sequence alignment modeling approach in the “Composer” module of

Sybyl 6.5 (Tripos Inc., St. Louis, MO). The experimentally-solved structures of

phenylalanine hydroxylase (1PHZ and 1PAH) (Kobe et al., 1999; Erlandsen et al.,

1997b) and tyrosine hydroxylase (1TOH) (Goodwill et al., 1997) were used as

template molecules in a modular method to provide the most complete model.

Initially, human TPH and rabbit TPH were predicted based on 1PHZ, 1PAH and

1TOH. This yielded initial structures with gaps at positions 1, 65 to 66, 100 to 101,

and 411 to 444 (Figure 7). The gaps that resulted within the protein (residues 65 to

66, and 100 to 101) were bridged using the “Loop Search” functionality in the Sybyl

biopolymer module. Before the loop search, each gap was enlarged by the removal

of two amino acids flanking the gap. Loop candidates were visually evaluated and

chosen on the basis of complementarity to the existing structure. In order to provide

a complete model, human and rabbit TPH were separately predicted based solely

on 1TOH and the carboxy terminus of the resulting structure (residues 411 to 444)

was then merged with the ir respective continuous, but truncated TPH model based

on 1PHZ, 1PAH, and 1TOH.

The resulting full-length human TPH model (missing only the initiator

methionine residue) was subjected to further refinement using the program

PROCHECK to assess progress (Laskowski et al., 1993). First, polar hydrogen

atoms were added, then proline and sidechain conformations were fixed and

73

relieved of bad contacts. Next, the peptide backbone was constrained and the

model was subjected to convergent minimization, without electrostatics, in the Tripos

forcefield (Clark et al., 1989). All the remaining valencies were hydrogen-filled and

the model was again minimized to convergence. Backbone constraints were then

removed and the model was minimized to a high level of convergence (gradient

0.005 kcal / mol⋅Å). Kollman charges (Weiner et al., 1984) were then added

followed by five cycles of minimization, during which time the dielectric constant was

gradually decreased from ε = 80 to ε = 20. The final minimization step was carried

out to convergence at a high level (gradient 0.005 kcal / mol⋅Å).

Bacterial expression of TPH

A full length cDNA clone for rabbit tryptophan hydroxylase has previously

been developed for prokaryotic expression (Vrana et al., 1993). The coding region

of the parent clone (prbTRH479) (Grenett et al., 1987) was amplified by PCR to

create a 5' NdeI site and a 3' BamHI site and subsequently subcloned into a pET-

21c prokaryotic expression vector. All recombinant proteins were expressed using

the approach detailed by Yohrling et al. (1999).

TPH deletion and point mutation constructs

Polymerase chain reaction was utilized to create a number of different

deletion mutations to examine TPH structure/function predictions from the human

TPH model. For example, to generate the N∆15 mutant (N ∆ denotes an amino

terminal deletion followed by the number of amino acid residues deleted from the

74

construct), a 5' primer was designed to introduce an NdeI consensus sequence

(CATATG) followed by the sequences for amino acid residues 16 to 20. Details for

the construction and characterization of the carboxy terminal mutant (C ∆17) are

outlined in (Mockus et al., 1997a). In addition, the S58A mutant utilized in this report

has been previously reported (Kumer et al., 1997). To elucidate a possible role for

S260 as a substrate for phosphorylation by CaMPKII, the mutants S260A and

S58A/S260A were created with the QuikChangeTM Site-Directed mutagenesis kit

from Stratagene. Wild-type TPH and S58A (25 ng) were used as the respective

DNA templates to create S260A and S58A/S260A. The conditions utilized are

outlined in (Yohrling et al., 1999; Vrana et al., 1994). The primers used (but not

previously reported) were as follows:

TPH N ∆15: 5'- CTCAATAGTCCATATGGGAAGAGCAAGTCTC -3'

TPH S260A Sense: 5'- GTGAGACACAGTGCAGACCCCTTCTATACC-3'

Antisense: 5'- GGTATAGAAGGGGTCTGCACTGTGTCTCAC -3'

TPH Y235A Sense: 5'- CGTCCTGTGGCTGGTGCCTTATCACCAAGAG -3'

Antisense: 5'- CTCTTGGTGATAAGGCACCAGCCACAGGACG -3'

TPH Y235L Sense: 5'- CGTCCTGTGGCTGGTCTCTTATCACCAAGAG-3'

Antisense: 5'- CTCTTGGTGATAAGAGACCAGCCACAGGACG -3'

The nucleotides underlined correspond to the NdeI recognition site. The bold

nucleotides highlight the mutated codons for amino acid residues 260 and 235,

respectively. Complete DNA sequencing was performed to verify the presence of

the appropriate mutation in the coding sequences of all recombinant proteins. This

also established that the PCR-based mutagenesis (N∆15) and non-PCR-based

75

mutagenesis (S260A, S58A/S260A, Y235A, and Y235L) did not introduce

extraneous mutations.

Kinetic analyses and TPH activity assay

Michaelis-Menten constants for two of the cosubstrates, BH4 and tryptophan,

were determined for wild-type TPH, TPH N ∆15, C ∆17, Y235A, and Y235L. First,

bacterial pellets containing the various proteins were sonicated in a HEPES-based

resuspension buffer and centrifuged at 50,000 x g to create a high speed

supernatant (Yohrling et al., 1999). The Km values of each recombinant protein for

BH4 were determined on comparable amounts of activity by performing a

radioenzymatic TPH activity assay with BH4 concentrations ranging from 9 µM -220

µM (Beevers et al., 1983; Vrana et al., 1993). Likewise, the Km of each enzyme for

tryptophan was calculated in the presence of tryptophan concentrations ranging

from 20 µM-1 mM. The activity values recorded at 400 µM and 1 mM of tryptophan

for wild-type TPH were not taken into account when determining the Michaelis

constant because of substrate inhibition. Stock solutions of L-tryptophan were

prepared in 200 mM Tris-HCl (pH 7.0). Activity values derived from each assay are

expressed as nmol product ⋅ hr-1 ⋅ mg total protein-1. All kinetic data were fitted to the

Michaelis-Menten equation using GraphPad Prism® version 3.0 (GraphPad

Software, San Diego, CA, www.graphpad.com).

76

Phosphorylation of native and mutant TPH

Bacterial supernatants of TPH and the mutants S58A, S260A, and

S58A/S260A were subjected to phosphorylating conditions to ascertain whether

S58A and/or S260 are phosphorylated by CaMPKII. Both amino acids reside in the

consensus motif for CaMPKII (Arg-X-X-Ser), and are predicted, by the hypothetical

model, to reside on the surface of the protein. Homogenates were incubated at

30°C for five minutes with 50 mM MgCl2, 3 mM CaCl2, 1µCi [γ-32P] ATP, 40 µM ATP,

0.75 µg calmodulin, and 400 units of CaMPKII. The reactions were terminated by

adding 5 µl of 6X sample buffer [0.5M Tris-HCl (pH 6.8), 30% (v/v) glycerol, 10%

(w/v) SDS, 0.6M DTT, and 0.012% bromophenol blue. The samples were

immediately denatured at 100°C for ten minutes and then subjected to denaturing

polyacrylamide gel electrophoresis (SDS-PAGE).

TPH protein analysis and enzyme activity

Denaturing polyacrylamide gel electrophoresis (10% acrylamide, 0.27% bis-

acrylamide) (Laemmli et al., 1970) was performed on phosphorylation reactions as

outlined by Kumer et al. (1997). Following transfer of the proteins, radioactive

proteins were visualized with a phosphoimager (Fuji Medical Systems).

Immunoreactive TPH proteins were detected by probing with a 1:1000 dilution of

affinity-purified mouse anti-TPH monoclonal antibody from mouse (WH3) followed

by a 1:1500 dilution (1.5 µg / ml) of sheep anti-mouse IgG coupled with HRP. The

incubation and wash conditions for western analysis are outlined in Mockus et al.

(1997b). Immune complexes were visualized using enhanced chemiluminescence

77

(NEN Research Products, Boston, MA) and exposed to X-ray film (Kodak Biomax

MR2) at exposure times (10 sec to 4 min) where the radioactivity signal was

negligent.

Acknowledgments

We would like to thank both Dr. Mark O. Lively (Wake Forest University) and

Dr. Susan M. Mockus (University of Washington) for their technical input. This work

was supported by PHS Grant RO1 GM-38931 to K.E.V.

78

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Res. 61, 313-320.

CHAPTER III

FUNCTIONAL ANALYSIS OF THE V177I CODING REGION POLYMORPHISM

IN HUMAN TRYPTOPHAN HYDROXYLASE (TPH1)

George C-T. Jiang, Amanda C. Domer, and Kent E. Vrana

The following manuscript is in preparation for submission to the Journal of

Neurochemistry. Stylistic variations are due to the requirements of this journal. The

original proposal and concept for these studies were those of George C.-T. Jiang.

Dr. Kent E. Vrana served in an advisory position on experimental design and

manuscript preparation. Amanda Domer assisted in the creation of point mutants,

protein expression and purification, and TPH activity assays.

82

83

Abstract

The aromatic amino acid hydroxylase (AAAH) superfamily consists of three

important enzymes: phenylalanine hydroxylase (PH), tyrosine hydroxylase (TH) and

tryptophan hydroxylase (TPH). These enzymes are rate-limiting in phenylalanine

catabolism, catecholamine biosynthesis, and serotonin biosynthesis, respectively.

Numerous polymorphisms in PH and TH have been described that have been

associated with phenylketonuria and dystonias, respectively, while few

polymorphisms have been described for TPH. Recently, the first human TPH coding

region polymorphism has been described. This paper analyzes the functional

implications of this polymorphism using site-directed mutagenesis, recombinant

bacterial expression, and purified enzyme. The Michaelis-Menten constant for

tetrahydrobiopterin was unchanged for V177I (Km, BH4 = 14.8 ± 0.9 µM), while the

enzyme’s Michaelis constant for the tryptophan substrate (Km, Trp = 41.1 ± 6.5 µM)

was increased 55% compared to wild-type enzyme (Km, BH4 = 15.8 ± 1.2 µM, Km, Trp

= 26.5 ± 3.3 µM). Our results indicate that the subtle V177I mutation results in a

somewhat less-efficient enzyme due to compromised tryptophan substrate binding.

Additional mutational analysis of V177 indicates that the identity of this residue

appears to be critical for proper enzyme function in that substitution of nearly any

amino acid at this position (except alanine, V177A) produces enzyme with little, or

no, activity. Therefore, while the specific polymorphism has a modest impact on

enzyme activity, it highlights a region of considerable importance to enzyme

structure and function.

84

Introduction

The aromatic amino acid hydroxylase (AAAH) superfamily is a group of

tetrahydrobiopterin-dependent monooxygenases with specificity for the benzene-

and indole-containing amino acids. The members of this family include

phenylalanine hydroxylase (PH; EC 1.14.16.1), tyrosine hydroxylase (TH; EC

1.14.16.2), and tryptophan hydroxylase (TPH; EC 1.14.16.4). These enzymes share

approximately 50% amino acid sequence identity and are thought to have evolved

from a common progenitor (Grenett et al., 1987; Neckameyer and White, 1992).

Each AAAH enzyme catalyzes the hydroxylation reaction of its respective amino

acid substrate in the presence of tetrahydrobiopterin (BH4) and molecular oxygen

(O2) co-substrates, and a ferrous iron (Fe2+) cofactor. The catalytic mechanisms of

the hydroxylases are believed to be sequential in nature with BH4 binding first and

forming part of the amino acid substrate-binding pocket, followed by binding of either

the molecular oxygen or the respective aromatic amino acid substrate (Fitzpatrick,

1991; Fitzpatrick, 1993; Andersen et al., 2002; Bassan et al., 2003). Recently,

Volner et al. (2003) have conclusively established that the mechanism for bacterial

PH is a fully-ordered ter-bi sequential mechanism.

The AAAHs are all composed of an amino terminal regulatory domain and a

carboxyl terminal catalytic domain (Grenett et al., 1987; Ledley et al., 1985; Darmon

et al., 1988; Daubner et al., 1993; Walker et al., 1994; Kumer et al., 1997). The

regulatory domains of the enzymes act to modulate activity. Documented

phosphorylation sites in all AAAHs reside in this regulatory domain (Kuhn et al.,

1997; Kumer et al., 1997; reviewed in Fitzpatrick, 2000). The active sites reside

85

within the catalytic domain and are responsible for the hydroxylation of their

respective aromatic amino acids. Chimeric hydroxylases have been created to

demonstrate that substrate specificity is conferred exclusively by the catalytic

domain (Mockus et al., 1997; Kumer et al., 1997). Crystal structures are available

for TH and PH (Goodwill et al., 1997; Goodwill et al., 1998; Erlandsen et al., 1997a;

Erlandsen et al., 1997b; Erlandsen et al., 1998; Fusetti et al., 1998; Erlandsen et al.,

2000; Erlandsen et al., 2002), while only one crystal structure is available for TPH

(Wang et al., 2002). The TPH crystal structure (Wang et al., 2002), hypothetical

AAAH models based on crystal structures (Jiang et al., 2000; McKinney et al., 2001;

Andersen et al., 2002), and NMR studies with TPH and bound substrates by

McKinney and colleagues (2001) predict to the residues involved in substrate

binding in TPH, and confirm the hypothesis of two binding pockets in the active site

– one for the aromatic amino acid substrate, and one for the tetrahydrobiopterin co-

substrate (BH4). These data are in agreement with the presumed catalytic

mechanisms of the AAAHs.

AAAHs have been implicated in several important clinical conditions. In the

case of PH, over 400 polymorphisms have been described that result in

phenylketonuria (PKU). A number of TH coding region polymorphisms have been

described that are associated with L-DOPA responsive parkinsonism (L205P,

Ludecke et al., 1996), dystonias (Q381K, Knappskog et al., 1995), and Segawa’s

Disease (Ludecke et al., 1995). However, due to the complexity involved in

diagnosing patients with serotonin aberrations, only one coding region

polymorphism has been described for TPH (Ramaekers et al., 2001). This

86

polymorphism is a heterozygous missense mutation within exon 6 (G529A) of the

TPH gene causing a substitution of isoleucine for valine at codon 177 (V177I). This

has been interpreted as a rare DNA variant because the pedigree analysis of that

individual did not provide direct genotype-phenotype correlation. This polymorphism

was discovered in a single member of a patient population that exhibited novel

neurodevelopment symptoms that were responsive to 5-hydroxytryptophan and

carbidopa treatment. In the other four patients, the TPH gene analysis was normal.

Notably, however, the individual exhibiting the TPH polymorphism also had another

commonly found polymorphism (C677T heterozygous mutation) in the

methylenetetrahydrofolate reductase (MTHFR) gene. The functional consequence

of this V177I mutation on TPH remains unclear. Moreover, because of the lack of

functional data, it is unclear if the serotonergic imbalances seen in the patients are

due to compromised TPH enzymes, inactivated TPH enzymes, or the selective loss

of serotonergic neurons. Structurally, we have localized the V177I polymorphism to

the catalytic domain of TPH, at a position near the tryptophan substrate-binding

pocket of the active site. Based on a TPH crystal structure (Wang et al., 2002) and

hypothetical TPH model (Jiang et al., 2000), Val177 is located approximately 3-4 Å

from Arg257 and approximately 5-6 Å from Ser336. These two residues are

conserved among the hydroxylases and have been implicated, within the other

AAAHs, in binding the alpha carboxyl group of the aromatic amino acid substrate

(Moran et al., 1998) and decoupling oxygen-oxygen bond cleavage and tyrosine

hydroxylation (Ellis et al., 2000), respectively.

To examine the functional significance of the TPH polymorphism, we used

87

site-directed mutagenesis to introduce this mutation into a recombinant human TPH

expression construct. The codon for valine 177 was altered to encode glycine,

alanine, isoleucine, leucine, phenylalanine, serine, threonine, arginine, and

glutamate. Mutant proteins were then expressed as hexa-histidine-tagged proteins,

purified, and assayed for tryptophan hydroxylation in order to determine the

biochemical consequences of the substitutions on TPH.

88

Materials and Methods

Materials

All materials were obtained from the Sigma Chemical Company (St. Louis,

MO) with the following exceptions: restriction and DNA modifying enzymes were

supplied by Promega Corporation (Madison, WI) and New England Biolabs (NEB;

Beverly, MA); L-[5-3H] tryptophan, and Ni2+ chelating columns from Amersham

Pharmacia Biotech (Piscataway, NJ); isopropyl-thio-β-D-galactopyranoside (IPTG),

ampicillin, and activated charcoal (Darco G-60) from Fisher Scientific (Pittsburgh,

PA). The pET-15b prokaryotic expression vector, and BL21(DE3)pLysS (F- OmpTrB-

mB-) competent cells were acquired from Novagen Corporation (Madison, WI).

Reinforced nitrocellulose (Duralose-UVTM) and the QuickChangeTM Site-Directed

Mutagenesis kit was purchased from Stratagene (La Jolla, CA). Benchmark

Prestained Protein Ladder, Mark 12™ Unstained Standard and Colloidal Blue

Staining Kit was purchased from Invitrogen (Carlsbad, CA). Chemiluminescence

reagents (RenaissanceTM) were purchased from NEN Research Products (Boston,

MA). The mouse anti-TPH monoclonal antibody (WH3) was obtained from Sigma-

RBI (St. Louis, MO). The sheep anti-mouse IgG HRP-coupled secondary antibody

was purchased from Amersham Life Sciences (Arlington Heights, Il). Qiagen

Miniprep and Hi-Speed Midiprep kits were purchased from Qiagen (Valencia, CA).

Finally, all sequencing was performed by the DNA Sequencing and Gene Analysis

Facility of the Molecular Genetics Program (Wake Forest University School of

Medicine) using a Perkin Elmer/Applied Biosystems 377 Prism automated DNA

89

sequencer.

TPH Point-Mutant Constructs

The methods used for the construction of the various TPH point-mutants are

similar to those outlined in Yohrling et al., 1998. The primers used are shown in

Table I. Briefly, mutations were made with the Stratagene QuikChangeTM Site-

Directed Mutagenesis kit using the pET-15b/hTPH plasmid as the template.

Thermocycler conditions were set as follows: 20 cycles of 94°C, 30s, 45°C, 30s,

72°C, 12m; 25 cycles of 94°C, 30s, 55°C, 30s, 72°C, 12m; 4°C. 2 µl DpnI-treated

products were used to transform supercompetent XL1-Blue cells. Random colonies

were selected to determine if the desired mutation was present. Complete DNA

sequencing was performed to verify the presence of the appropriate mutation in the

coding sequences of all recombinant proteins and to discount the presence of

spurious sequence mutations.

90

Table I – Site-directed mutagenesis primers

h-V177Is 5’- G ACC TGG GGA GCC ATT TTC CAA GAG C -3’

h-V177Ia 5’- G CTC TTG GAA AAT GGC TCC CCA GGT C-3’

h-V177As 5’- G ACC TGG GGA GCC GCA TTC CAA GAG C -3’

h-V177Aa 5’- G CTC TTG GAA TGC GGC TCC CCA GGT C-3’

h-V177Rs 5’- G ACC TGG GGA GCC CGA TTC CAA GAG C -3’

h-V177Ra 5’- G CTC TTG GAA TCG GGC TCC CCA GGT C-3’

h-V177Es 5’- G ACC TGG GGA GCC GAA TTC CAA GAG C -3’

h-V177Ea 5’- G CTC TTG GAA TTC GGC TCC CCA GGT C-3’

h-V177Ls 5’- G ACC TGG GGA GCC CTT TTC CAA GAG C -3’

h-V177La 5’- G CTC TTG GAA AAG GGC TCC CCA GGT C-3’

h-V177Fs 5’- G ACC TGG GGA GCC TTT TTC CAA GAG C -3’

h-V177Fa 5’- G CTC TTG GAA AAA GGC TCC CCA GGT C-3’

h-V177Ga 5’- G ACC TGG GGA GCC GGT TTC CAA GAG C -3

h-V177Ga 5’- G CTC TTG GAA ACC GGC TCC CCA GGT C-3

h-V177Ss 5’- G ACC TGG GGA GCC TCT TTC CAA GAG C -3’

h-V177Sa 5’- G CTC TTG GAA AGA GGC TCC CCA GGT C-3’

h-V177Ts 5’- G ACC TGG GGA GCC ACT TTC CAA GAG C -3’

h-V177Ta 5’- G CTC TTG GAA AGT GGC TCC CCA GGT C-3’

Bolded nucleotides corresponding to the codon which yields an altered amino acid.

91

Protein Expression and Purification of Human TPH

Human tryptophan hydroxylase (hTPH) cDNA was a generous gift of Dr.

Jacques Mallet (Centre National de la Recherche Scientifique, Paris, France).

Based on the recent report of Walther et al. (2003), this is the peripherally-

expressed isoform of the enzyme and might more accurately be described as TPH1.

PCR primers integrating NdeI and BamHI sites were utilized to modify the

respective 5’ and 3’ ends of the hTPH gene to enable subcloning into pET

prokaryotic expression vectors (Novagen, Inc., Madison, WI). PCR products were

digested with both restriction enzymes and then ligated into a similarly digested

pET-15b expression vector that encodes an amino-terminal hexa-histidine tag.

Clones were verified by complete DNA sequencing. This sequence analysis

confirmed clone identity and eliminates any extraneous mutations that may have

occurred during the PCR modification step.

All recombinant proteins were expressed using the approach detailed in

(Yohrling et al., 1999). Briefly, recombinant human TPH (hTPH) was expressed in

bacteria by transforming the BL21(DE3)pLysS strain of Escherichia coli (E. coli).

Bacteria were grown until they reached mid-log phase as determined by a

spectrophotometer reading of OD600 = 0.4. Bacteria were then induced by the

addition of isopropyl-thio-β-D-galactopyranoside (IPTG) to express hTPH for 2.5

hours at 30°C. Bacterial cells were harvested by low speed centrifugation.

Recovery of recombinant protein was achieved by lysis of the cells via sonication in

a HEPES-based resuspension buffer, centrifugation at 50,000 x g, and subsequent

collection of the supernatant.

92

The clarified lysate (high speed supernatant) was then subject to a one-step

metal chelate affinity chromatography procedure using an AktaFPLC automated

workstation (Amersham Pharmacia Biotech, Piscataway, NJ) and an optimized

protocol for the HisTrap Kit (Amerham Pharmacia Biotech, Piscataway, NJ). Our

modifications to the purification protocol include the addition of 100mM imidazole in

the start and wash buffers, and elution of the His-tagged protein in a buffer

containing 300mM imidazole, all performed on a 5ml Ni2+-chelating column at a flow

rate of 3ml/min. Purified protein was immediately assayed or stored at –80°C in a

storage buffer consisting of 50mM HEPES (pH 7.5), 0.2M NaCl, 10% glycerol,

0.05% Tween 20, and 1mM dithiothreitol and protease inhibitors (Hamdan and

Ribeiro, 1999).

Kinetic Analyses and TPH activity assay

TPH enzyme activity was determined using a radioenzymatic assay as

previously described with substrate concentrations of 100 µM BH4 and 50 µM

tryptophan (Beevers et al., 1983; Vrana et al., 1993; Jiang et al., 2000). Michaelis-

Menten constants for two of the co-substrates, BH4 and tryptophan, were

determined for wild-type hTPH, and the hTPH V177 mutants. The Km values of

each recombinant protein for BH4 were determined on comparable amounts of

activity by performing a radioenzymatic TPH activity assay with BH4 concentrations

ranging from 6-110 µM (at 50 µM tryptophan) (Beevers et al., 1983; Vrana et al.,

1993; Jiang et al., 2000). Likewise, the Km of each enzyme for tryptophan was

calculated in the presence of tryptophan concentrations ranging from 2µM-2mM (at

93

100 µM BH4) (Jiang et al., 2000). Activity values, derived from each assay, are

expressed as nmol H2O product • min-1 • mg-1. Because 5-hydroxytryptophan

production is equimolar with water production, the nmol of product are considered

identical. All kinetic data were fitted to the Michaelis-Menten equation using

GraphPad Prism version 3.03 (GraphPad Software, San Diego, CA,

www.graphpad.com).

94

Results

Expression and purification of wild-type and mutant enzymes was not

significantly different (Table II and Figure 2). All active enzymes demonstrated

similar half-lives and stability (activity retention) in storage buffer (data not shown),

though initial baseline activity may have been different (Table II and III). The

V177G, V177S, V177T, V177R, V177E, V177L and V177F mutants demonstrated

significantly compromised enzyme activity compared to the wild-type enzyme, with

the V177R and V177E mutants having no detectable activity.

The experimental kinetic data for wild-type and active mutant enzymes are

shown in Table III. Wild-type TPH exhibited a Michaelis-Menten constant for

tetrahydrobiopterin (Km, BH4) of 15.8 ± 1.2 µM, and a Km, Trp = 26.5 ± 3.3 µM. The

mutant V177I, which is the polymorphism described by Ramaekers and colleagues

(2002), showed no change with respect to its Michaelis-Menten constant for

tetrahydrobiopterin (Km, BH4 = 14.8 ± 0.9 µM), but demonstrated an apparent

decrease in Vmax when assayed for its Km, BH4 at a fixed tryptophan concentration of

50 µM. This apparent decrease in Vmax was not observed when varying

concentrations of BH4 were assayed with a higher tryptophan substrate

concentration (500 µM). Conversely, the Michaelis-Menten constant for the actual

tryptophan substrate increased (Km, Trp = 41.1 ± 6.5 µM) with no significant change

in Vmax. Similarly, the V177A mutant did not exhibit a change in Km, BH4 (16.7 ± 2.5

µM) or in Km, Trp (31.6 ± 1.4 µM) with no significant changes in Vmax. Kinetic

observations for V177G, V177S, V177T, V177L, V177F, V177R, and V177E were

95

unable to be determined as all of these mutants demonstrated little or no activitiy

following purification (<5% of wild-type activity).

96

Discussion

There is considerable similarity among the AAAH in terms of sequence

identity, overall structure and catalytic reaction. Therefore, it has been suggested

that the TPH active site is comprised of two distinct binding regions – one for

tetrahydrobiopterin and one for tryptophan – and that TPH exhibits a comparable

catalytic mechanism to the other AAAH. Namely, TPH would utilize an ordered ter-

bi sequential binding order with tetrahydrobiopterin binding first followed by either

oxygen or tryptophan (Fitzpatrick, 1991; Fitzpatrick, 1993; Andersen et al., 2002;

Bassan et al., 2003; Volner et al., 2003). This mechanism, along with the

hypothetical TPH models (Jiang et al., 2000; McKinney et al., 2001; Martinez et al.,

2001) and TPH crystal structure (Wang et al., 2002), assist in interpreting the

experimental data obtained with the V177 mutants. Specifically, our hypothetical

model of TPH (Jiang et al., 2000), predictions based on NMR data (McKinney et al.,

2001), and x-ray crystallography data (Wang et al., 2002) suggest a structural

situation presented in Figure 1. Namely, that arginine 257 (R257) is actively

engaged in orienting binding of the alpha-carboxyl of the tryptophan substrate, as in

TH (Moran et al., 1998). While not directly involved in substrate binding, V177 is

close enough to R257 (3-4 Å) to influence its orientation.

Replacement of the valine residue with a slightly larger isoleucine residue

mimics the coding region polymorphism of TPH (Ramaekers et al., 2001), and

results in a decrease in Vmax (observed at lower fixed concentrations of tryptophan

substrate) with no change in Km, BH4. However, this subtle mutation seems to hinder

the binding and/or utilization of the tryptophan substrate as demonstrated by the

97

increase in Km, Trp with no change in Vmax. This change can be explained by the

steric hindrance provided by the bulkier isoleucine residue to either the substrate

itself or the position of the surrounding residues. Further support for this hypothesis

is given by the data obtained from analysis of the valine to alanine substitution at

residue 177 (V177A), which did not adversely affect the performance of the enzyme

with respect to BH4 or tryptophan. However, a further reduction in side chain size

and hydrophobicity, through construction of the V177G point mutant, is detrimental

to enzymatic activity. In fact, this mutation nearly abolished enzyme activity. This

can be explained by the lack of any steric hindrance, thus resulting in conformational

flexibility that could dramatically perturb the size and shape of the active site pocket.

Similarly, V177S and V177T, mutants exhibiting side chains similar in size to

alanine and valine, respectively, but with hydrophilic properties, also exhibited little

enzyme activity.

Unexpectedly, the V177L mutant also had little enzyme activity. Our

hypothesis was that this mutation would result in an enzyme that had activity with no

change in Km, BH4, and an increase in the Km, Trp compared to V177I. Though

typically considered a relatively conservative mutation, it is plausible that the

addition of a methylene group could constrain the positioning of residue 177 and

shift the orientation of surrounding residues (such as R257), thereby preventing

optimal residue placement and decreasing normal enzyme function. In contrast, the

lack of activity of the V177F mutant was expected as we hypothesized that the bulk

and hydrophobicity of the phenylalanine side chain would perturb the local structure

and/or proper binding of the tryptophan substrate to such a great extent that the

98

enzyme would not be able to function. Together, these mutagenesis experiments

provide further support for a theory that this residue plays a critical, but indirect, role

in the proper positioning of the tryptophan substrate.

Replacement of the valine residue with charged residues, namely arginine

and glutamate, resulted in point mutants that had no detectable activity. These

results from V177R and V177E are not unexpected as these residues introduce a

charge into the hydrophobic core of the enzyme, which could easily perturb local

structure and thereby prevent proper binding and subsequent catalysis. Structural

models of these mutants allowed for theoretical visualization of the residue at

position 177 potentially interacting with the nearby arginine 257 residue (3-4Å). In

the case of V177R, the close proximity of the side chains would result in Van der

Waals repulsive forces, while a potential salt bridge could form between V177E and

R257, all of which would perturb proper tryptophan substrate binding if R257 is

indeed involved in coordinating the alpha carboxyl carbon of the amino acid

substrate as in TH (Moran et al., 1998).

Typically, a valine to isoleucine substitution is considered a subtle change.

Our data indicate that this mutation at residue 177 (V177I) is not completely

deleterious to TPH enzyme activity, and suggest that this polymorphism could exist

in the population but remain undiagnosed as long as tryptophan substrate levels are

high. Physiologically, such a mutation would exhibit itself only in the presence of an

additional metabolism-related defect. This coincides with the findings of Ramaekers

and colleagues, where only one out of the 5 patients that exhibited aberrant

serotonin neurochemistry and the neurodevelopment problems possessed the TPH

99

polymorphism. Overall, our data give credence to the theory that the decrease in

serotonin levels in these neurodevelopmentally-challenged patients results from a

loss in serotonergic neurons rather than a decrease in serotonin levels due to a

change in tryptophan hydroxylase activity.

Complicating the explanation of the potential functional role of the V177I

mutation is the recent discovery of a brain-specific isoform of TPH gene, Tph2

(Walther et al., 2003). This novel isoform, Tph2, encodes a TPH enzyme that

predominates in the central nervous system, and resembles the TPH enzyme

encoded by Tph1 (Boularand et al., 1990; Boularand et al., 1995), studied in

Ramaekers et al. (2002), and reportedly to be located in the periphery (Walther et

al., 2003).

It is logical to assume that the neuronal-specific TPH (TPH2, Walther et al.,

2003) plays the dominant role in the regulation of normal neurological development

and function. Based on the fact that the V177I polymorphism was observed in only

1 out of 5 patients, coupled with the recent report that the TPH harboring this

polymorphism is the peripheral enzyme, it is likely that V177I is coincidentally

associated with the novel neurodevelopmental disorder. It is important to note,

however, that the presence of this polymorphism highlighted a residue which has

proven to be pivotally important to the structure of the active site. Without evidence

of this polymorphism, there would have been no reason a priori to select valine 177

for mutational analysis. Fully understanding this neurodevelopmental disorder will

have to await the characterization of TPH2 in these patients, as well as an

understanding of its interrelationship with TPH1. It seems unlikely that a

100

polymorphism in the peripheral version of TPH would not play some role in a

behavioral syndrome that is corrected with treatment with 5-hydroxytryptophan and

carbidopa supplementation and is characterized by reduced serotonin metabolites.

Acknowledgments

We would like to thank Dr. Jacques Mallet (Centre National de la Recherche

Scientifique, Paris, France) for the generous gift of human tryptophan hydroxylase

(hTPH1). We would like to thank Dr. Linda C. McPhail, Dr. Mark O. Lively, Dr.

Leslie B. Poole, and Dr. William E. Sonntag (Wake Forest University) for their

technical input. This work was supported by PHS grant R01 GM-38931 (to K.E.V.).

101

Figure 1 – Structural depictions of Val177 mutations. A closeup view of the active

site site pocket of the hypothetical model of human TPH is depicted (Jiang et al.,

2000). Secondary structure is identified by color: β-sheets (yellow), α-helix (pink),

and random coil (white). The locations of Tyr235, Arg257, Ser260, and Ser336 are

indicated. A) The typical active site conformation and positioning of Val177;

Predicted structural changes resulting from V177 substitutions are represented on

the hypothetical TPH model in B) Val177Gly; C) Val177Ser; D) Val177Thr; E)

Val177Ala; F) Val177Ile; G) Val177Leu; H) Val177Phe; I) Val177Arg; J)

Val177Glu. These images were generated on a Silicon Graphics Workstation with

the modeling program Sybyl 6.8 (Tripos Inc., St. Louis, MO).

102

A

B C D

E F G

H I J

103

Table II - Purification data for wild-type and mutant enzymes Protein Yield (µg) a Specific Activity b Fold purification c

Wild-type 160 21.9 661

V177G 155 0.41 120

V177S 165 0.66 2.88

V177T 175 1.04 447

V177A 150 44.4 500

V177I 154 28.6 3920

V177L 180 0.99 102

V177F 171 ND -

V177R 140 ND -

V177E 106 ND -

a Yield of purified protein from 250ml of bacterial culture (average 25 mg total protein).

b Units of specific activity were nmol 5-OH-Trp produced / min-mg. TPH enzymatic

activity was determined (see Materials and Methods) using substrate concentrations of

100 µM BH4 and 50 µM tryptophan.

c Fold purification was determined using specific activity values determined from enzyme

activity in the crude bacterial extract and purified fraction.

ND – No detectable activity.

104

Figure 2 - Coomassie and immunoblots of purified proteins. Human TPH was

expressed in E. coli (BL21) as an amino-terminal oligohistidine fusion protein and

purified to apparent homogeneity by affinity chromatography. 1 µg purified protein

was subject to SDS-PAGE. (A) Colloidal Blue staining of purified TPH proteins.

Lane L: Mark 12™ Unstained Standard; lane1: V177G; lane 2: V177S; lane 3:

V177T; lane 4: V177A; lane 5: wild-type TPH; lane 6: V177I; lane 7: V177L; lane 8:

V177F; lane 9: V177R; lane 10: V177E; lane 11: 1 µg BSA; lane 12: 5 µg BSA; lane

13: 10µg BSA. (B) Immunoblot of purified TPH proteins. Lane1: V177G; lane 2:

V177S; lane 3: V177T; lane 4: V177A; lane 5: wild-type TPH; lane 6: V177I; lane 7:

V177L; lane 8: V177F; lane 9: V177R; lane 10: V177E. Immunoreactive proteins

were detected with a monoclonal antibody (WH3) at a 1:1000 dilution followed by an

anti-mouse IgG-HRP from sheep at a 1:1500 dilution. Each protein migrated with an

apparent molecular mass of 51 kDa.

105

106

Table III - Steady-state kinetic parameters of wild-type TPH and mutants

Enzyme Km, BH4 (µM) Km, Trp (µM) Specific Activity (nmol/min/mg)

Wild-type 15.8 ± 1.2 26.4 ± 3.3 21.9

V177A 16.7 ± 2.5 31.6 ± 1.4 44.4

V177I 14.8 ± 0.9 41.1 ± 6.5 * 28.6

V177G NR - 0.41

V177S NR - 0.66

V177T NR - 1.04

V177L - - 0.99

V177F - - 0.02

V177R - - -

V177E - - -

NR – Not Reliable. Activity levels were too low to provide accurate Km determinations, but

values ranged from (17.5 µM to 55 µM). Cells denoted with “-“ provided no kinetic values.

TPH activity was determined (see Materials and Methods) and the kinetic constants were

calculated by direct fit to the Michaelis-Menten equation and non-linear regression analysis

using GraphPad Prism version 3.03 (GraphPad Software, San Diego, CA,

www.graphpad.com). Kinetic determinations used substrate concentrations at 100 µM BH4

(tryptophan variable) and 50 µM or 500 µM tryptophan (BH4 variable). The mean values for

BH4 and tryptophan were compared independently of each other (n = 6 to 11). Specific

activity was determined at substrate concentrations of 100 µM BH4 and 50 µM tryptophan.

*One-way ANOVA, followed by a Student’s Newman-Keuls test at 95% confidence indicated

that the kinetic differences observed were statistically significant (p = 0.004).

107

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Vrana S.L., Dworkin S.I., and Vrana K.E. (1993) Radioenzymatic assay for

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terminal sequences contributing to tryptophan hydroxylase tetramer formation. J. Mol. Neurosci. 12, 23-34.

CHAPTER IV

SUMMARY AND CONCLUSIONS

George C.-T. Jiang

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Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the

formation of the neurotransmitter serotonin (5-HT, Grahame-Smith, 1964;

Lovenberg et al., 1967), and also plays a role in the subsequent biosynthesis of

melatonin (Chanut et al., 2002; Privat et al., 2002; reviewed in Martinez et al., 2001).

TPH is localized predominantly within the raphe nuclei of the midbrain and is also

peripherally located in the pineal gland, retina, and enteric neurons of the gut

(Jequier et al., 1969; Joh et al., 1975; Austin and O’Donnell, 1999; reviewed in

Mockus and Vrana, 1998). Recent studies reveal that this may be explained by the

existence of two distinct isoforms of TPH that are differentially expressed in the CNS

and periphery (Walther et al., 2003). Serotonin, and therefore TPH by inference,

play important roles in a variety of physiological processes. Serotonin is implicated

in a variety of CNS functions such as temperature control, aggression, pain and

memory (Kandasamy and Williams, 1984; reviewed in Oliver et al., 1995; Brentegani

and Lico, 1982; Cornwell-Jones et al., 1989). Aberrations in serotonin synthesis,

regulation, and homeostasis have been associated with numerous psychiatric

illnesses including depression, anxiety, obsessive-compulsive disorder (OCD),

bulimia, migraines, and drug abuse (reviewed in Mockus and Vrana, 1998).

The present work has encompassed the development of a hypothetical TPH

model (Jiang et al., 2000), studies to validate the model and identify amino acid

residues of importance (Jiang et al., 2000), and studies that address the significance

of a TPH polymorphism found in one individual (Jiang et al., in preparation) (see

Figure 1). The hypothetical model has served as an excellent tool to visualize

structure and function relationships, and aided in mutagenesis studies, in lieu of a

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TPH crystal structure that was only recently released (Wang et al., 2002). The

recent publication and release of the TPH crystal structure definitively illustrates the

molecular structure in the catalytic domain and further validates the hypothetical

model, and the experimental studies conducted in this work. An “iterative magic fit”

of the TPH crystal structure and hypothetical model using Swiss-PdbViewer (Guex

and Peitsch, 1997) indicate a root mean square deviation (RMSD) of 1.02 Å

between the carbon backbones of the structures. This demonstates the high degree

of similarity between both structures considering rms deviations between models of

homologous protein structures and real ones are typically between 2 and 4 Å, even

in the best cases (Petsko, 2000).

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Figure 1 – Structural location of the functional residues of interest in the present

work. A view of the hypothetical model of human TPH is depicted (Jiang et al.,

2000). Secondary structure is identified by color: β-sheets (yellow), α-helix (pink),

and random coil (white). Residues of note include Ser58, Val177, Tyr235, Ser260,

and Ser443. This image was generated on a Silicon Graphics Workstation with the

modeling program Sybyl 6.8 (Tripos Inc., St. Louis, MO).

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The present work has identified CaMPKII phosphorylation sites in TPH. The

substrate amino acid residues include Ser58, Ser260, and Ser443. Mutagenesis

studies with Ser58Ala and Ser260Ala mutants indicate that phosphorylation can

occur at either residue and only minimally at Ser443. We surmise that Ser443 is

only a potential CaMPKII phosphorylation site, and probably plays a minimal role in

in vivo regulation of TPH as it is located in the extreme carboxy terminus

tetramerization domain of the enzyme and would therefore normally be buried deep

within the protein. The small amount of phosphorylation that was observed in the

double point mutant (Ser58Ala/Ser260Ala) was, in all probability, a result of the

presence of monomeric or denatured TPH in solution.

The development of a hypothetical TPH structure has been useful in further

understanding structure/function relationships in this important biosynthetic enzyme

by assisting in the selection of amino acid residues for mutagenesis studies.

Mutagenesis experiments and subsequent characterization have identified and

confirmed the importance of Val177 and Tyr235 in the catalytic domain of TPH.

Site-directed mutagenesis of Val177 allowed the correlation of functional data for a

naturally-occurring coding region polymorphism associated with a developmentally-

impaired patient (Ramaekers et al., 2001). Our in vitro studies with recombinantly-

expressed human TPH indicate that Val177 plays a role in tryptophan substrate

utilization and the identity of the residue is critical for proper enzyme structure and

function (Jiang et al., in preparation). Similarly, mutagenesis studies on Tyr235

reveal that the residue plays an integral role in the BH4 binding pocket that

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dramatically affects subsequent tryptophan hydroxylation (Jiang et al., 2000;

McKinney et al., 2001; Wang et al., 2002; reviewed in Martinez et al., 2001).

Recently, a genetic knock-out of the known TPH gene was created in mice

(Walther et al., 2003). Interestingly, the knock-out mice (Tph-/-) still had normal

serotonin (5-HT) levels in classically serotonergic brain regions in the central

nervous system, while they lacked 5-HT in the periphery. This led the authors to

identify a new isoform of TPH (Tph2) in mice, and subsequently in rat and human

species. The knockout mice (Tph1-/-) exhibited no significant behavioral differences

in elevated plus maze and hole board tests, which are indicative of 5-HT-related

behaviors. These data from mice are different from the C. elegans TPH knockout

that was previously generated (Sze et al., 2000). These TPH deletion mutant worms

were viable, but did not synthesize serotonin, and exhibited alterations in their

behavior and metabolism, including decreased feeding and egg laying rates, and an

increase in fat storage (Sze et al., 2000). Comparisons between the amino acid

sequences of Tph1, Tph2, and C. elegans Tph reveal that the bulk of the differences

lie in the amino terminal regulatory domain, but do not identify the C. elegans TPH

gene more closely with either of the mammalian genes.

The recent identification of the new TPH isoform in vertebrates, which

predominates in the central nervous system, raises interesting new questions

regarding the regulation of serotonin biosynthesis in animals, and the possibility for

potential intervention with novel pharmacotherapies specific for CNS TPH or

peripheral TPH. This could have dramatic implications for future treatment of

serotonergic disorders. One can envision using genetic therapies by engineering the

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necessary TPH gene to either increase or decrease the protein’s inherent enzyme

stability or perhaps its basal activity. The full-length human TPH model and crystal

structure have been useful in filling some of the gaps in our knowledge of the

structure, function, and regulation of TPH, and will continue to do so in the fuure.

In conclusion, the AAAH are highly homologous proteins, but subtle nuances

exist between the enzymes that determine substrate specificity, stability, and

enzyme regulation. Though the short half-life of TPH and minute quantity available

in a single animal have historically impeded progress on the research of TPH, these

challenges are slowly being conquered. The recent advances in protein expression

technologies has helped to overcome problems with enzyme purification, while the

identification of a stable form of TPH in Schistosoma mansoni (Hamdan and Ribeiro,

1999) has provided new insights to TPH enzyme stability and regulation. Moreover,

the development of a hypothetical TPH structure and the subsequent crystallization

provide new tools to further expand the knowledge base regarding TPH structure

and function. It is hoped that information from this thesis, and future studies using

the contemporary tools and approaches available will assist in addressing the

important issues and intricate details that remain about serotonin biosynthesis and

TPH regulation in human health and disease.

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Grahame-Smith, D.G. (1964) Tryptophan hydroxylation in brain. Biochem Biophys

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Hamdan, F.F., and Ribeiro P. (1999) Characterization of a stable form of tryptophan

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Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin. Biochem. 41, 12569-12574.

APPENDIX

PHENYLKETONURIA

George C-T. Jiang, George J. Yohrling IV, and Kent E. Vrana

The following educational review article is in press for publication in the

Encyclopedia of Life Sciences and is reprinted with permission from the Nature

Publishing Group, www.els.net. Stylistic variations are due to the requirements of

this journal. The manuscript was prepared and edited by George C.-T. Jiang.

Authorship is shared with George J. Yohrling IV and Dr. Kent E. Vrana. George J.

Yohrling IV wrote the initial draft of the Future Treatments for Phenylketonuria

section. Dr. Kent E. Vrana served as a scientific advisor/editor for this project.

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125

Introduction

Metabolic disorders can adversely affect the body’s natural homeostatic or

steady state and lead to chemical imbalances and severe pathological conditions.

Phenylketonuria is such an example in which the normal conversion of the dietary

amino acid phenylalanine to tyrosine is blocked. The resulting build-up of

phenylalanine and its metabolites in young patients produces a number of severe

side effects including intellectual impairment and cutaneous changes. This article

reviews the autosomal recessive metabolic disorder of phenylketonuria, covering

clinical diagnoses, typical and atypical causes, as well as current and potential

treatments.

Phenylketonuria, also known as PKU, is a disorder that affects 1 in 12,000–

15,000 births. In most cases, the cause is a deficiency in the enzyme phenylalanine

hydroxylase (PH). This genetic condition results in numerous complications due to

the incomplete metabolism of the essential amino acid phenylalanine. Clinical

manifestations, if the disease is left untreated, include developmental delay and

severe intellectual impairment, seizures, autism, eczema, hyperactivity, aggressive

behaviour and scleroderma-like skin changes with dilution of hair and skin colour.

PKU is implicated in about 0.64% of institutionalized patients worldwide. Early

diagnosis after birth is critical and treatment with a phenylalanine-restricted diet will

prevent onset of intellectual impairment and neurological abnormalities. Routine

paediatric screening for PKU began in the United States in 1961, and the success of

screening has spurred the development of similar procedures for other genetic

metabolic disorders.

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Clinical Signs and Conditions

PKU is caused by a deficiency in the enzyme PH or reduction in the synthesis

or regeneration of a critical co-substrate for the enzyme. In patients afflicted with

PKU, this results in a build-up of phenylalanine, as it is not converted to tyrosine.

Collectively, the various forms of PKU are also referred to as hyperphenylalaninemia

owing to the increased circulating levels of the amino acid. The excess

phenylalanine is metabolized to abnormal breakdown products (the phenylketones)

accounting for the common name of the disorder, phenylketonuria. Increased levels

of phenylalanine and its metabolites cause irreparable damage to the developing

central nervous system unless the condition is diagnosed and treated within the first

3-6 weeks of life. Furthermore, this biochemical defect can result in a variety of

cutaneous abnormalities, including diffuse hypopigmentation, eczema, and

photosensitivity. These cutaneous changes may result from the toxic effects of

phenylalanine and its decomposition products in the skin (Figure 1).

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Figure 1 Phenylalanine metabolism. Phenylalanine hydroxylase (PH) catalyzes the

conversion of phenylalanine to tyrosine. Deficiencies in the activity of this enzyme

result in incomplete phenylalanine metabolism and build-up of toxic waste products.

BH

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129

Normally, the essential amino acid phenylalanine is converted to tyrosine by

the enzyme PH. The resulting tyrosine can then be converted, via independent

pathways, to catecholamines (the neurotransmitters dopamine, noradrenaline

(norepinephrine) and adrenaline (epinephrine)), melanin, or thyroid hormone. In

addition, tyrosine can be incorporated, as a fundamental building block, into new

proteins. Because tyrosine is itself a nonessential amino acid, PKU is not fatal since

a diet high in tyrosine can make up for the lack of PH activity. Reduced synthesis of

the cosubstrate tetrahydrobiopterin, or a deficiency in the regenerating enzyme

dihydropteridine reductase may also result in phenylketonuria. However, such

cases are rarer and are referred to as non-classical PKU.

With the aid of routine paediatric screening, a diagnosis of PKU is usually

established soon after birth. The newborn screening test that is performed involves

the measurement of blood phenylalanine levels by the Guthrie test, a bacterial

inhibition assay. Normally, blood phenylalanine levels are below 2mg/dL-1. A blood

phenylalanine concentration of greater than 20mg/dL-1 with a normal or reduced

concentration of tyrosine is diagnostic of classical PKU. For the confirmation of

PKU, all amino acid levels must be measured by a quantitative technique and

screening programmes often request repeat specimens in any infant with a blood

phenylalanine level greater than normal.

The essential neurological symptoms are intellectual impairment, which may

cause corresponding changes in electroencephalogram, disturbed gait, ataxia,

extrapyramidal symptoms, focalized or generalized epileptiform seizures. In

general, if treatment of PKU is instituted by 3 weeks of age, prevention of intellectual

130

impairment and neurologic abnormalities is seen. However, learning disabilities

have been reported in even well-treated patients. Today, it is recommended that

patients with PKU continue a phenylalanine-restricted diet indefinitely because of

several reports of decreased intellectual ability in children who were taken off the

diet at 5 or 6 years of age. Continuation of the diet also aids in prevention of

complications that are seen in genetically normal infants of women with PKU

(maternal PKU). These complications include microcephaly, congenital heart

disease and intrauterine growth delay.

In patients with PKU, cutaneous changes can result from the toxic effects of

phenylalanine and its decomposition products in the skin. Changes include

reduction of skin, hair, and eye colour with increased photosensitivity, although

photosensitivity is not universal. Dermatitis is also seen in up to 50% of patients.

This dermatitis is virtually indistinguishable from the true atopic dermatitis, a chronic

skin condition characterised by red itchy skin. Some are indeed true cases, but

others only mimic such lesions. Hair and skin darkening and other skin change

reversals may occur with dietary restriction of phenylalanine intake. However, when

a diet with normal levels of phenylalanine is resumed, facial eczema may reappear

within 24 h.

The current treatment for patients afflicted with PKU is a strict dietary regimen

that is restricted in phenylalanine intake yet provides all the other amino acids. The

earlier the treatment is started, the more beneficial the effects. While on dietary

treatment, patients with PKU need to be wary of products that contain the artificial

sweetener aspartame (Nutrasweet). Aspartame, chemically known as N-

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aspartylphenylalanine methyl ester, is commonly used in sweetened drinks, and the

amount in a litre of sweetened drink can be equivalent to the amount of

phenylalanine normally obtained from the daily diet. Uninformed patients may

therefore be at risk for contradicting their phenylalanine-reduced diet.

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Classical Phenylketonuria

Typically, the term classical PKU refers to hyperphenylalaninemias resulting

from a deficient PH enzyme. If left untreated, classical PKU can lead to severe

intellectual impairment. As stated previously, PH is the mixed function oxidase

which, in its central reaction mechanism, converts the essential amino acid L-

phenylalanine to L-tyrosine in the presence of the co-substrates tetrahydrobiopterin

and molecular oxygen. The recognition that PH was the cause of the

hyperphenylalaninemia was not made until the late 1940s, over 20 years after

classical PKU was initially characterised by Asbjörn Fölling in 1934. It is important

to note that PKU does not necessarily result from a lack of the entire PH gene.

Assays to detect the PH protein show that patients with PKU can produce the

enzyme and kinetic studies provide evidence for enzymatic deficiencies.

The human PH gene was first cloned in 1985; since then many researchers

have studied it to determine the molecular basis for PKU. The PH gene comprises

13 exons and spans approximately 90 kilobases on the human chromosomes.

Many different defects in the PH gene have been observed and reported for patients

with PKU. These polymorphisms include single nucleotide base changes or point

mutations, deletions, splice variants, premature stop signals and insertions. Most of

the identified polymorphisms are single nucleotide changes that result in a protein

with one amino acid substituted by another. These missense mutations constitute

approximately 60% of all the known mutations that have been characterized.

Splicing mutations and deletions each constitute about 13% of all the mutations. In

these cases, a mutation results in defective ribonucleic acid processing (splicing).

133

This can produce an absence of PH or a defective form of the enzyme. Nonsense

mutations (inappropriate protein synthesis stop signals) and insertions make up the

remainder of the polymorphisms. The majority of the mutations (approximately

80%) lie in the central catalytic domain of the protein between exons 5 and 12, but

mutations have been seen in all the different exons. Table 1 lists the various causes

of PKU.

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Table 1 Genetic defects and substrate deficiencies in phenylketonuria.

135

136

The frequencies at which different populations exhibit distinct polymorphisms vary

greatly. Caucasians tend to have a mutation in the splice site of intron 12 where an

adenosine is substituted for a guanosine (G→A), resulting in a truncated form of PH

that is missing the last 52 amino acids. Asians, on the other hand, do not have such

a general genotype. Based on these types of geographic localizations, it is clear

that PKU has evolved from numerous independent mutations.

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Nonclassical Phenylketonuria

In addition to the classical forms of PKU involving mutations in the PH gene,

there are a number of examples of the disease (1-3% of the PKU cases) where the

underlying defect is in other systems. Notably, these involve mutations in the

dihydropteridine reductase (DHPR), or in the biosynthetic enzymes responsible for

biopterin synthesis (see Figure 1). For this reason, neonatal patients exhibiting

hyperphenylalaninemia must also be tested for urinary pteridines, blood DHPR, and

additional biogenic amines. This last test is necessary because defects in

tetrahydrobiopterin (BH4) synthesis or regeneration (from quinonoid

dihydrobiopterin; q-BH2) will affect other key biosynthetic enzymes, including

tyrosine hydroxylase (rate-limiting enzyme in dopamine, noradrenaline and

adrenaline biosynthesis) and tryptophan hydroxylase (rate-limiting enzyme in

serotonin biosynthesis). Unfortunately, these patients are subject to a much more

severe prognosis than those with classical PKU. Disruption of these other

neurotransmitter systems produces mental health problems and motor problems

(similar to Parkinson disease).

Infants with nonclassical PKU (pterin cosubstrate deficiencies) will require

additional therapeutic interventions. First, they require limitations in dietary

phenylalanine to prevent the build-up of the amino acid and its phenylketone

metabolites. However, this will further exacerbate the deficiency in the

catecholamine neurotransmitters (dopamine, noradrenaline and adrenaline).

Therefore, patients with biopterin defects are also treated with precursors to the

catecholamines that bypass the tyrosine hydroxylase step (L-dopamine; the same

139

Future Treatments for Phenylketonuria

As stated above, the current therapeutic regimen for the majority of the

patients with PKU is the dietary restriction of the amino acid, phenylalanine. This

can reduce the blood levels of phenylalanine and help prevent the severe intellectual

impairment common with this disease. Regardless of the successes with this

intervention, there are problems with this approach. Dietary restriction is often

difficult and lends itself to non-compliance. This is particularly true in those cases

where dietary phenylalanine restriction is recommended for life. It has been

reported, for instance, that a decline in intellectual function and behavioural

performance is evident in adults with PKU who have not maintained a low

phenylalanine diet.

Dietary management is particularly important in pregnant women with

classical PKU who have previously curtailed phenylalanine restriction. Dietary non-

compliance during pregnancy can produce PKU-like symptoms in their genetically

unaffected (heterozygous) offspring. This syndrome is referred to as “maternal

PKU”. In addition to the intellectual impairments of their offspring, low birth weight

and congenital heart disease are often present. To prevent this, dietary treatment

must be commenced before the pregnancy even begins, and rigidly maintained for

the duration of the pregnancy.

While dietary treatment for PKU is effective and non-hazardous, additional

developments in the field are needed in order to find a “cure” for PKU. There are

several different mechanisms that are amenable to clinical intervention. For

example, drugs could be designed that would prevent phenylalanine absorption from

140

the gut. This would cause ingested phenylalanine to be excreted and may prevent

its damaging effects. Enzyme therapies may also be utilized. Insertion of active PH

into a “fatty” liposome carrier or administration of regular enzyme injections may

serve potential roles in the treatment of PKU.

Additional attention needs to be paid to PH, the primary enzyme in the

metabolism of phenylalanine. As stated above, PH hydroxylates phenylalanine in

the presence of the necessary substrates to form the amino acid tyrosine.

Numerous defects in the gene encoding PH lead to an inactive enzyme and the

systemic build-up of phenylalanine in the body. The recent X-ray crystallographic

analysis of PH structure has supplied valuable information regarding the structure of

the protein. These data may assist researchers in the design of

pharmacotherapeutic interventions to treat PKU by accelerating the metabolism of

phenylalanine to tyrosine. Further structural and functional studies, such as these,

are necessary if we hope to eradicate PKU completely.

Recently, researchers using rodent models found that use of oral enzyme

therapy, with the enzyme phenylalanine ammonia lyase (PAL), combined with a low

protein diet has the potential to replace the traditional phenylalanine-restricted

dietary regimen. PAL is an enzyme that does not require a cofactor to convert

phenylalanine to trans-cinnamic acid and insignificant amounts of ammonia. Trans-

cinnamic acid is a harmless metabolite that is further converted to benzoic acid and

then to hippurate which is excreted in urine. In theory, patients on PAL could break

down phenylalanine such that phenylalanine ingestion could be tolerated. In rodent

models, it was found that oral administration of PAL attenuated PKU. When

141

humans were used as test subjects, similar results were observed. However, only

limited data are available from the studies because of the costs necessary to obtain

sufficient amounts of PAL. Further research must be performed before the general

population can use PAL, but these studies indicate that oral administration of PAL

appears to have great potential as a future treatment for PKU. The costs of

acquiring PAL must decrease however.

Research indicates that PKU is primarily the result of single gene

malfunctions. Therefore, a promising alternative to dietary restriction of

phenylalanine in the management of PKU is somatic gene therapy. This would

involve the insertion of a normal PH gene into a chromosomal location in the

nucleus of liver cells (hepatocytes). The normal PH would then be expressed in

place of, or in addition to, the mutated PH. Hepatocytes would be the appropriate

targets for gene therapy since PH is known to be expressed predominantly within

the liver.

In order to get the normal gene to its proper target, the deoxyribonucleic acid

(DNA) needs to be inserted into a vector or “carrier” system. To date, the use of

three different vector systems has been explored. They include recombinant

retroviral vectors, recombinant adenoviral vectors, and DNA-protein complexes.

Numerous studies have shown these gene carriers to be successful in lowering

blood phenylalanine levels. However, all have serious limitations, such as low

transduction efficiencies and the inability to integrate into non-dividing cells.

Additionally, these vectors may eventually lead to immune responses, or become

inactive, which limits long-term efficacy. All of these limitations must first be

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overcome if genetic therapy for PKU is to become a reality.

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Summary

Phenylketonuria (hyperphenylalaninemia) is one of the more common genetic

disorders of intermediary metabolism. Disruption of the normal metabolic

conversion of phenylalanine to tyrosine results in increased circulating levels of

phenylalanine and its metabolic breakdown products. In the developing neonate,

these metabolic imbalances produce dramatic clinical problems including severe

intellectual impairment. In most cases, the genetic culprit in this problem is a defect

in the biosynthetic enzyme PH. In a minority of the cases, there is an alternative

problem in the biosynthesis or regeneration of a pivotal co-substrate of the PH

reaction (BH4). Luckily, if properly diagnosed soon after birth, PKU can be managed

through dietary interventions that decrease the levels of circulating phenylalanine.

Knowing the molecular biology of the disorder provides a long-term hope of “curing”

the disease through gene replacement strategies. These approaches may well

become routinely available in the future.

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Further Reading

Bremer, H.J., Duran, D., Kamerling, J.P., Przyrembel, H., Wadman, S.K. (1981) Clinical Chemistry and Diagnosis of Inherited Diseases. Disturbances of Amino Acid Metabolism: Clinical Chemistry and Diagnosis. pp. 307-327. Baltimore: Urban & Scwharzenberg. [Chapter on clinical chemistry and diagnosis of inherited diseases.]

Eisensmith R.C. and Woo, S.L.C. (1995) Molecular Genetics of Phenylketonuria:

From Molecular Anthropology to Gene Therapy. Advances in Genetics 32, 199-271.

Hoang L., Byck S., Prevost L., Scriver C.R. (1996) PAH Mutation Analysis

Consortium Database: a database for disease-producing and other allelic variation at the human PAH locus. Nucleic Acids Research, 24(1):127-131.

Kaufman, S. (1997) Tetrahydrobiopterin. pp. 262-322. Baltimore: John Hopkins

University Press. [Chapter on phenylketonuria and its variants.] Nowacki P.M., Byck S., Prevost L., Scriver C.R. (1997) The PAH Mutation Analysis

Consortium Database Update 1996. Nucleic Acids Research 25(1):139-142. Wurtman, R. and Ritter-Walker, E. (1988) Dietary Phenylalanine and Brain Function.

Boston, MA: Birkhäuser.

CURRICULUM VITA

George Chih-Thai Jiang

BORN: June 12, 1978 Bangkok, Thailand

EDUCATION: Wake Forest University School of Medicine

Winston-Salem, North Carolina M.S., Molecular Genetics, 2003 Wake Forest University Winston-Salem, North Carolina M.B.A., 2003

University of Maryland, College Park College Park, Maryland B.S., General Biology, 1998

SCHOLASTIC AND PROFESSIONAL EXPERIENCE:

Howard Hughes Undergraduate Research Fellow, Department of Microbiology and Imuunology, University of Maryland, College Park, College Park, MD, 1997-1998. Advisor: Dr. Elisabeth Gantt Research Assistant, Department of Plant Biology, University of Maryland, College Park, College Park, MD, 1994-1997. Advisor: Dr. Elisabeth Gantt Research Assistant, Department of Gastroenterology, Walter Reed Army Institute of Research, Washington, D.C., 1994-1995. Advisor: Dr Yuan-Heng Tai

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146

HONORS and AWARDS:

Dean’s Fellowship, Wake Forest University Graduate School of Arts and Sciences, 1998-1999. Bacock Scholarship, Wake Forest University Babcock School of Management, 2001-2002. 2nd Place Graduate Student Poster Award, Western North Carolina Society for Neuroscience, 2001.

PROFESSIONAL APPOINTMENTS AND ACTIVITIES:

Graduate School Activities

Volunteer, Community Public Affairs Committee, 1999-present Representative, Graduate Student Association, 1999-present President, Graduate Student Association, 2001-2002 Member, Wake Forest University Graduate Council, 2001-2002

Graduate Teaching Experience

Laboratory Instructor – Recombinant DNA

Technologies, 1999, 2000 Course Coordinator – Drug Discovery and Development, 2002

Ad hoc reviewer: Journal of Biological Chemistry

Journal of Genetics and Development Journal of Neurochemistry Nature Structural Biology Proceedings of the National Academy of Sciences

Structure

PROFESSIONAL MEMBERSHIPS:

Society for Neuroscience, 1999-present. Western North Carolina Society for Neuroscience, 2000-present. American Society for Biochemistry and Molecular Biology, 2000-present. National Association of Graduate Professional Students, 2001-present.

147

BIBLIOGRAPHY:

JOURNAL ARTICLES:

Yohrling IV GJ, Jiang GC-T, Mockus SM, and Vrana KE (2000). Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase. J. Neurosci. Res. 61(3): 313-320.

Jiang GC-T, Yohrling IV GJ, Schmitt JD, and Vrana KE (2000). Identification of

substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling. J. Mol. Biol. 302(4): 1005-1017.

Blanchard-Fillion B, Souza JM, Friel T, Jiang GC-T, Vrana KE, Sharov V, Barón L,

Schöneich C, Quijano C, Alvarez B, Radi R, Przedborski S, Fernando GS, Horwitz J, and Ischiropoulos H (2001). Nitration and Inactivation of Tyrosine Hydroxylase by Peroxynitrite. J. Biol. Chem. 276: 46017-46023.

Yohrling IV GJ, Jiang GC-T, DeJohn MM, Robertson DJ, Vrana KE, Cha JH-J

(2002). Inhibition of Tryptophan Hydroxylase Activity and Decreased 5-HT1A Receptor Binding in a Mouse Model of Huntington’s Disease. J. Neurochem. 82:1416-1423.

Stredrick DL, Jiang GC-T, Suber L, Mockus S, Aschner M, and Vrana KE.

Manganese-Mediated Inhibition of Tyrosine Hydroxylase in CATH.a Cells. Mol. Brain Res. (in revision).

Yohrling IV GJ, Jiang GC-T, DeJohn MM, Miller DW, Young AB, Vrana KE, Cha JH-

J. Analysis of Cellular, Transgenic and Human Models of Huntington’s Disease Reveals Tyrosine Hydroxylase Alterations in Substantia Nigra Neuropathology. J. Neurosci. (submitted).

Jiang GC-T, Domer AC, and Vrana KE. Functional Analysis of the V177I Coding

Region Polymorphism in Human Tryptophan Hydroxylase (TPH1). (in preparation).

BOOK CHAPTERS: Jiang GC-T, Yohrling IV GJ, and Vrana KE. (March 2000). Phenylketonuria. In:

Encyclopedia of Life Sciences. Vol. 14, pp. 150-154. London: Nature-Scientific American Publishing Group. http://www.els.net (invited review).

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Yohrling IV GJ, Jiang GC-T, and Vrana KE. (August 2000). Control of Enzymatic

Activity. In: Encyclopedia of Life Sciences. London: Nature-Scientific American Publishing Group. http://www.els.net (invited review).

ABSTRACTS: Yohrling IV GJ, Jiang GC-T, Mockus SM, and Vrana KE. Site-directed

mutagenesis of tryptophan hydroxylase reveals K111-E223 to be an intersubunit dimerization bridge. Soc. Neurosci. Abst. 25: 178, 1999.

Jiang GC-T, Yohrling IV GJ, Schmitt JD, and Vrana KE. Molecular modeling of

human tryptophan hydroxylase: Implications for subunit assembly and drug development. FASEB J. 14(8): A1453, 2000.

Yohrling IV GJ, Jiang, GC-T, Schmitt, JD, and Vrana KE. Identification of a

tryptophan orienting residue in the active site of tryptophan hydroxylase. Soc. Neurosci. Abst. 26: 2000.

Robertson DJ, Allen JW, Freeman WM, Jiang GC-T, Walker SJ, Hyyta P, Kiianmaa

K, Sampson HH and Vrana KE. Changes in VTA gene expression following ethanol self-administration in AA/ANA rats. RSA Abst. 2000.

Yohrling IV GJ, Jiang GC-T, Schmitt JD, and Vrana KE. Identification of a

tryptophan orienting residue in the active site of tryptophan hydroxylase. Soc. Neurosci. Abst. 26:400, 2000.

Jiang GC-T, Freeman WM, Vrana KE, Caron MG, and Jones SR. Functional

neurogenomics and biochemical analyses of enzymatic differences in dopamine transporter knockout mice. Soc. Neurosci. Abst. 27, 2001.

Yohrling IV GJ, Jiang GC-T, Robertson DJ, Vrana KE, and Cha J-HJ. Biochemical

Analyses of Tyrosine and Tryptophan Hydroxylase in the R6/2 Mouse Model of Huntington’s Disease. Soc. Neurosci. Abst. 27, 2001.

Jiang GC-T, Yohrling IV GJ, Miller DW, DeJohn MM, Vrana KE, and Cha J-HJ.

Tyrosine hydroxylase activity and mRNA changes in Huntington’s disease. Soc. Neurosci. Abst. 28, 2002.