Chapter 1

INTRODUCTION

Chapter1

Tuberculosis (TB) is one ofthe leading infectious diseases and affects roughly one-third of

the entire world population (Sander et al., 2004). A total of 1.6 million people died ofTB,

including 1,95,000 patients infected with HIV (WHO, 2007). Mycobacterium tuberculosis

is the etiological agent of the disease which most commonly affects the lungs. In healthy

people, infection with the pathogen often causes no symptoms, since the person's immune

system acts to "wall off' the bacteria. The pathogenicity of M. tuberculosis results from its

potential to multiply to high parasite burdens and its unique capability to modulate the

host immune system. In view of the annual death toll of approximately 2 million

individuals due to infection by M. tuberculosis, the emergence of multi drug-resistant

(MDR) and extensively drug-resistant (XDR) strains of M tuberculosis poses a major

threat to human health worldwide (http://www.who.int). Treatment of this disease is

further complicated by the ability of M. tuberculosis to persist in the lungs of infected

individuals for decades by switching to a dormant or latent phase (Bloom et al., 1999)

which also induces tolerance to current antibiotics (Wayne, 1994 & Wallis et al., 1999).

About one-third of the world's populations are infected with persistent mycobacteria,

providing an enormous potential reservoir for further spread of this disease. Dormancy has

been associated with nonreplicating or very slow growth of M. tuberculosis that resides in

granulomas, a heterogeneous assembly of macrophages, in the lungs of infected

individuals. It is generally assumed that the microenvironment in the granulomas is

characterized by hypoxia, nutrient starvation, presence of reactive oxygen and nitrogen

species (Wayne, 1994; Zhang, 2004 & Fenton et al., 1996)).

The availability of complete genome sequence has enhanced our capability to

understand molecular basis of disease and understand the complex host-pathogen

interactions. The sequencing of its genome revealed that it is comprised of 4,411 ,529 base

pairs which code for -4000 genes. M. tuberculosis differs radically from other bacteria in

that a significant portion of its coding capacity is devoted to the production of enzymes

involved in lipogenesis and lipolysis (Cole et ai., 1998). Analysis of the sequences have

resulted in attributing precise functions to 40% of the predicted proteins, some information

is available for 44% and the remaining 16% might account for specific mycobacterial

functions (Cole et al., 1998). A re-annotation suggests that the genome contains 3995

genes coding for proteins (ORPs) and 50 RNA genes (45 tRNA, 3 rRNA and 2 stable

rRNA) (Cole et ai., 1998 & Camus et ai., 2002).

Chapter1

Coordinated efforts have been launched by several TB Structural Genomics

Consortiums (http://www.doe-mbi.ucla.edu/TB) to elucidate the crystal structures of

proteins from the pathogen. These efforts are expected to greatly contribute to the

identification of novel drug targets and the development of new inhibitors with therapeutic

potential aided by rational methods.

1.1 PLP Dependent Enzymes Various enzymes require a co-factor to carry out the reaction. ego NAD+ and

F AD+ co-factors are used by dehydrogenases to carry out various oxidation-reduction

reactions. Other examples of co-factors include biotin in decarboxylation reactions, heme

in 02 transport and chlorophyll for photosynthesis. These coenzymes bind covalently or

non-covalently with their respective apoenzymes. Pyridoxal phosphate (PLP; a vitamin B6

derivative) arguably represents the most versatile organic co-factor in biology (John, 1995;

Jansonius, 1998; Mehta et al., 2000 & Christen et al., 2001). It acts as a coenzyme in a

vast number of reactions in amino acid metabolism. In many free living prokaryotes

almost 1.5% of all genes codes for PLP-dependent enzymes, but in higher eukaryotes the

percentage is substantially lower, consistent with these catalysts being involved mainly in

basic metabolism (Percudani et al., 2003). PLP was identified in 1951 as one of the active

vitamers of vitamin B6 (Heyl, 1951), PLP (Fig 1.1) has subsequently been extensively

researched to understand the basis of its catalytic versatility.

PLP-dependent enzymes are unrivaled in the diversity of reactions that they

catalyze (Scheme 1.1) (Eliot et al., 2004). All enzymes that use PLP as a co-factor are

associated with biochemical pathways that involve amino compounds, mainly amino

acids, except glycogen phosphorylase (Palm et al., 1990) which uses PLP in quite a

different way (John, 1995). The protein moiety of a given B6 enzyme determines which of

the many potential pathways is adopted by the coenzyme-substrate adduct. Furthermore,

PLP-dependent catalysis is dominated by a sequence of steps involving different

intermediates, some of which are common to most of the members of this enzyme family

(Scheme 1.1). Normally in the native enzyme, PLP is bound via a Schiff base to an active

site lysine and forms an internal aldimine. In the presence of substrate or substrate

analogues, catalysis proceeds via formation of gem-diamine, external aldimine, quinonoid,

ketimine or aminoacrylate intermediates (Mozzarelli et al., 2006).

2

Fig 1.1: Schematic diagram Pyridoxal S' phosphate (PLP)

Y-Synthase

~J;.,,~S9 "' r,.~ cr

Aminotransferases .... _-­Ii' to C4

IT Ii' from Ca

.. ........ .. _- .. -... ".

Decarboxylases

~~ • External Aldimioe .~

"" .. ,,' .. ... .......... _---- --_. " .. -

H+to Ql - Ii ·Synthase

:~~P'~~'~~-;;';':;t;d ': _ A .Eliminase : aldimine: .., ....... .... -...................... ..

~ ... ~ a -Synthase

~ ('0 ~ ~ J;." U c~

Chapter1

/ ,,"" /.~ ~

Transaminating Decarboxy .. ses

~v~~ , •••• •• • _ • • _ • •••••••••• • <#-\~4>~

~:' loteroalAldimioe \ ~ . . ~0 Sor;"" Hydro. ymothyl . . . . .. .... ,~ .. .... _-.. ... _ ... _,-_ .. . Q> tranafen.e

Scheme 1.1: Schematic representation of the onglDs of different reaction specificities. Reactions catalysed by all these enzymes begin with conversion of internal to external aldimine. The natures of the covalent transfonnations occurring at successive steps are indicated by arrows connecting the intennediates. Intennediates designated Q are quinonoid in character. (Figure is adapted from John, 1995)

3

Chapter]

1.2 Mechanistic versatility of PLP as a co-factor A large number of reactions carried out by PLP dependent enzymes involve amino

acids. These include the transfer of the amino group, decarboxylation, interconversion of

L- and D-amino acids, and removal (elimination) or replacement of chemical groups

bound at the (3- or "1- carbon (Scheme 1.1). Such versatility arises from the ability of PLP

to covalently bind to the substrate and then to function as an electrophilic catalyst, thereby

stabilizing different types of carbanionic reaction intermediates (John, 1995; Schneider et

al., 2000). The co-factor in all cases functions to stabilize the negative charge

development at Cex in the transition state that is formed after condensation of amino acid

substrate with PLP to form a Schiff base (Stryer, 1995). The stabilization of the Cexanion is

facilitated by delocalization of the negative charge through the pi system of the co-factor,

and for this reason PLP is often described as an electron sink (Christen et al., 2001).

The co-factor has two basic chemical properties; in the first instance through its

aldehyde group, its forms an imine with the amino groups of substrates. In the second

instance because it acts as an electron sink, it withdraws electrons from the substrate

(John, 1995). Initially the aldehyde group ofPLP is covalently linked to a lysine residue at

the active site to form an internal aldimine. Upon binding of the amino acid substrate, the

lysine is exchanged for the amino group of substrate forming a Schiff base complex with

PLP (external aldimine). Its 5' -phosphate group is attached to the protein matrix through

up to nine hydrogen bonds and is also often stabilized by charge interactions (Jansonius,

1998). In the next step of reaction, one of the bonds to the Cex atom of the external aldimine

is broken resulting in the formation of the quinonoid intermediate (Hayashi, 1995). This

process is facilitated by the electrophilic properties of the co-factor, which can act as

electron sink and stabilize the developing negative charge. The magnitudes of reactions

catalyzed by PLP-dependent enzymes have several steps in common and variations arise

from enzymatic control of different routes to the central quinonoid intermediate (i.e.

depending upon which of three groups bound to Ca atom of the substrate is cleaved off).

An important basis for the reaction specificity of B6 enzymes seems to be the orientation

of bonds at Cex of the substrate moiety in the external aldimine adduct. Together with the

Ccx-N bond, the bond to be broken is supposed to lie in a plane orthogonal to the plane of

the coenzyme-imine 7r system of pyridine ring imine system (Dunathan, 1966 & Dunathan,

1971).

4

Chapter1

1.3 Structural diversity of PLP dependent enzymes Many structures of Vitamin B6 dependent enzymes are now available. The first

classification of PLP dependent enzymes was suggested by Alexander and co-worker

(Alexander et at., 1994) who divided PLP-dependent enzymes into Ct, (3 and 'Y classes

depending on the carbon atom involved in the chemistry. Goldsmith and colleagues

(Grishin et aI., 1995) classified PLP dependent enzymes into five different fold types

on the basis of amino acid sequence comparison, predicted secondary structure

elements and 3D structural information. All PLP enzymes whose structures have been

solved to date can be classified into one of the five fold types (Table 1.1). Two

additional fold types, VI and VII, were tentatively introduced to classify some

enzymes of unknown structure. Cartoon representations of the structures of a

representative of each fold type are in Fig 1.2.

1.3.1 Fold type I (Aspartate aminotransferase family) enzymes The enzymes in this class are active as a homodimer or high order oligomers

with two active sites per dimer. Each subunit folds into two domains, a large domain in

which the central feature is a seven stranded (3 sheet, and a small domain, comprising

the C-terminal part of the chain, which folds into a three or four stranded (3 sheet

flanked by helices on one side (Fig 1.2A). The active site is located in a cleft between

the two domains, at the interface between the two subunits of dimer. Residues from

both domains and both subunits are involved in co-factor binding (Schneider et at., 2000). In general the two active sites are independent but asymmetry has been

observed in a few cases (Churchich et at., 1981 & Hennig et al., 1997). However, two

structural features have a corresponding identifiable signature within the sequences of

fold type I enzymes. First, the Schiff base lysine is closer to the C-terminus than the

glycine-rich region and second, an invariant aspartic acid, which binds to the pyridoxal

ring nitrogen atom (pyridine nitrogen), precedes the Schiff base lysine by 20-50 amino

acids (Grishin et al., 1995).

An analysis of pair-wise structurally aligned enzymes provided a basis for

evaluation of evolutionary relationships within this fold type. This analysis suggested

that fold type I enzyme can be further divided into six subclasses, of which three

correspond to aminotransferases (Table 1.2) (Grishin et al., 1995 & Mehta et at.,

1993).

5

Chapter1

1.3.2 Fold II (Tryptophan synthase ~) family The structure of fold type II (tryptophan synthase, TRPS family) enzymes is

similar to those of fold I except that members of this fold type are evolutionarily

distinct (Mehta et al., 2000). This family includes the trytophan synthase {3 subunit

(Hyde et al., 1988), threonine deaminase (Gallagher et al., 1998) and o-acetyl

serinesulfhydrylase (Burkhard et al., 1998). The TRPS exz{3z is a dimer of two fold­

related pairs, in which {3 subunits lie back to back (Fig 1.2B). The two dimer (cx{3) act

independently, but each is allosterically regulated through its neighbouring subunit in

the dimer, such that substrate binding to the ex-subunit enhances the {3-reaction and vice

versa. The ex-subunit which has a TIM-barrel fold act as a regulatory unit, while the {3

subunit (C-terminal domain) is a PLP dependent enzyme and contains two domains

comprised of a mixed cxJ{3 structure with PLP bound between these two domains (Hyde

et al., 1988). The PLP binding regions in these enzymes share the same fold and

consist of two domains of similar size; an N-terminal domain containing a four

stranded sheet surrounded by helices and a C-terminal domain build up by six stranded

~l1CCl witll fllHlleing lleHee~. In tlle enzyme i1 ~crine rc~idue euurdini1te:3 the pyridine

nitrogen, rather than the aspartic acid observed in the aspartate aminotransferase

family.

1.3.3 Fold III (Alanine racemase) family Alanine racemase (Shaw et al., 1997) which is representative member of this

fold is a homodimer where each of the subunits folds into two domains, an eight­

stranded cxJ{3 barrel and a domain mainly comprising {3 strands (Fig 1.2C). PLP binds at

the mouth of the cxJ{3 barrel in a cleft formed between the two domains. Contrary to the

PLP dependent enzyme classes described above, PLP binds with the re side facing the

protein. The side chain of an arginine residue forms a hydrogen bond to the pyridine

nitrogen, indicating that the protonation of the pyridine nitrogen is not crucial for

catalysis in these enzymes (Eswaramoorthy et al., 2003). Further, all enzymes of this

family are obligate dimers, as each monomer contributes residues to both active sites

(Kern et al., 1999).

6

1.~.4 Fold IV (f)-amino acid aminotransferase) family This class presently has only two members with known 3D structures; D-amino

acid aminotransferase (Sugio et ai., 1995) and branched-chain aminotransferase

(Okada et ai., 1997). The active site of this class of proteins is virtually a mirror image

of the active site of fold type I aminotransferases (Sugio et ai., 1995). The fold

consists of two-domain structure with the active site located at domain interface (Fig

1.2D). Two identical subunit forms a catalytically competent dimer, however branched

chain aminotransferase further assemble into a hexamer. The active site lysine is

situated in a loop between the first strand in the barrel and a following helix. The

phosphate group of PLP is bound to the N-terminus of an a-helix in the same domain.

The co-factor binds with its re side facing the protein rather than active site pocket as

observed in the fold type I family, accounting for the difference in stereochemistry of

the products in the reaction of D-amino acid aminotransferase. A glutamic acid side

chain forms a hydrogen bond to the pyridine nitrogen.

1.3.5 Fold V (Glycogen phosphorylase) In members of this class of enzymes, PLP does not act as an electrophilic catalyst

but instead its phosphate group participates in transfer. However, because glycogen

phosphorylase binds PLP in a very specific manner and PLP is used for catalytic purposes

this enzyme can be considered to belong to the superfamily of vitamin B6 dependent

enzymes (Weber et ai., 1978 & Sprang et ai., 1979). Glycogen phosphorylase is multi

domain protein whose C-terminal domain has a dinucleotide binding fold and bind with

the co-factor PLP. The phosphate group binds to the N-termius of a helix which also

harbors the active site lysine residue one tum further down the helix. No hydrogen bonds

are made from protein to pyridinium nitrogen in this enzyme.

7

Chapter]

(A) (B)

(C) (D)

Fig 1.2: Ribbon diagram of representative enzymes of fold I - IV. Each structure depicts a monomer. PLP is shown in blue. (A). Fold type I (E. coli aminotransferase, PDB file IASN). (B). Fold Type II (Salmonella typhimurium O-acetyl serinesulfhydrylase; 10AS). (C). Fold type ill (Bacillus stearothermophilus, Alanine racemase; ISFT). (D). Fold type IV (Thermophilic Bacillus Spp. D-aminoacid aminotransferase; IDAA)

8

Chapter1

Table 1.1: The super family of vitamin B6 dependent enzyme.

Fold type Enzyme Source Family PDB

References code

Fold Type I

Aminotransferase Aspartate Chicken a 7AAT McPhalen subclass I aminotransferase mitochondria et al., 1992

Chicken cytosol a 2CST Malashkevich et al., 1995

Pig cytosol a 1AAT Malashkevich et al., 1982

Escherichia coli a lARS Okamoto et al., 1994

Saccharomyces a 1YAA Jeffery et al., cerevisiae 1998 Thermus a 1BJW Nakai et al., 1999 thermophilus

Aromatic Paracoccus a 1AY4 Okamoto et al., aminotransferase denitrificans 1998 Tyrosine Trypanosoma cruzi a 1BWO Blankenfeldt et aminotransferase al., 1999

Escherichia coli a 3TAT Ko et al., 1999 Aminotransferase GABA- Pig a 10HV Storici et al., 2004 subclass II aminotransferase

Escherichia coli a 1SF2 Liu et al., 2004

Ornithine Homo sapiens a 10AT Shen et al., 19998 aminotransferase

Dialkylglycine Pseudomonas a 2DKB Toney et at., decarboxylase cepacia 1993 Glutamate-1- Synechoccoccus sp. a 2GSA Shell et al., 1997 semialdehyde aminomutase Acetyl ornithine T. thermophilus 1VEF aminotransferase

Daminopelargonate Escherichia coli a 1QJ5 Kack et al., 1999 synthase

Aminotransferase Phosphoserine Escheria. coli a 1BJN Hennig et al., subclass IV aminotransferase 1999

Bacillus a 1BT4 alcalol!.hilus

Fold type II

Tryptophan synthase Salmonella {3 1UBS Rhee et at., {3 subunit typhimurium 1997 O-acetylserine Salmonella {3 10AS Burkhard et at., sulphydrylase typhimurium 1998 Threonine deaminase Escherichia coli {3 lTDJ Gallagher et at.,

2004

9

Chapter1

Fold type III

Alanine racemase Bacillus lSFT Shaw et at., stearothermophilus 1997

Eukaryotic ornithine mouse 70De Kern et al., 1999 decarboxy lase Yeast hypothetical Saccharomyces IB54 Eswaramoorthy protein cerevisiae et al., 2003

Fold type IV

D-Amino acid Bacillus sphaericus lAOG Sugio et at., aminotransferase 1995 Branched-chain Escherichia coli lA3G Okada et al., 1997 amino acid aminotransferase

Human lEKF Yennawar et al., 2001

Fold type V

Glycogen Oryctolagus lA8I Gregoriou et phosphorylase cunicuius 1998 Maltodextrin Escherichia coli lAHP O'Reilly et phosphorylase 1997

1.4 Aminotransferases Aminotransferases (ATs) (EC 2.6.2.x) belongs to the a-family of vitamin B6 -

dependent enzymes. These enzymes play important roles in amino acid biosynthesis,

degradation and carbohydrate metabolism in both prokaryotes and eukaryotes. ATs have

several properties like there is no requirement of external addition of co-factor, broad

substrate specificity, high enantioselectivity and regioselectivity, high reaction rate and

stability (Taylor et at., 1998). ATs play an important role in the metabolism of amino

acids of all species by catalyzing the reversible transfer of an amino group from amino

acid substrate to an acceptor oxo acid. With other PLP dependent enzymes they share

certain mechanistic features like covalent bond formation of an aldimine between

pyridoxal-P and the amino acid substrate as the first intermediate (Christen, 1985). Each

PLP dependent enzyme operates by a mechanism, common to all aminotransferases, in

which the co-factor shuttles between pyridoxal phosphate bound via a Schiff base and

non-covalently bound pyridoxamine forms by means of two coupled half reactions

(Scheme 1.2). The second half reaction involving the conversion of an a-keto acid to (X-

10

al.,

al.,

Chapter1

amino acid is almost common for most aminotransferases. Since the aminotransferase

reaction requires two different substrates to bind to the same co-factor active site, the

enzyme must be able to accommodate both substrates in the active site while

discriminating between them in the different reaction steps. All aminotransferases known

to date use the same coenzyme, PLP, to catalyze the same type of reaction and are

distinguished mainly by their substrate specificity. Majority of known structures of fold

type I enzymes, invariably function as a homo dimer or as a higher order oligomer with

two active sites per dimer. The active site lies on the dimeric interface and each monomer

contributes essential residues to both active sites. In all ATs, the PLP co-factor is

positioned in the active site by numerous non-covalent interactions so that si face of

pyridine ring lies towards the incoming substrate (Hwang et al., 2005).

Aminotransferases, which form one of the largest groups among PLP enzymes,

are subdivided into four subclasses. The enzymes in the first three classes belong to

fold type I while group III aminotransferases belong to fold IV of the PLP dependent

enzyme family (Table 1.2). This classification correlates with the structure of N­

terminal part of the chain which folds similarly within a subclass but shows entirely

different conformations between the subclasses (Mehta et al., 1993).

1.4.1 Group I aminotransferases Members of group I aminotransferases appear to be more versatile than other

members of aminotransferase family because its members accept alanine, dicarboxylic and

aromatic amino acids as substrates (Mehta et al., 1993). This subclass of

aminotransferases can be further divided into two subgroups based on the reaction

specificities (Table 1.2). Aspartate aminotransferase (AspAT) (Okamoto et al., 1994) and

aromatic aminotransferase (ArAT) from Paracoccus denitrificans (Okamoto et al., 1998) are

the most widely studied ATs belong to group Ia while methionine aminotransferase

(MetAT) (Dolzan et al., 2004), histidinol phosphate aminotransferase (HspAT)

(Haruyama et al., 2001), tyrosine aminotransferase (TAT) (Blankenfeldt et al., 1999),

glutamine: phenylpyruvate aminotransferase (GlnAT) (Goto et al., 2004), ArAT from

Pyrococcus horikoshii (Matsui et al., 2000) and kynurenine aminotransferase (KAT) (Rossi et

al., 2004) belong to group lb. All these ATs are homodimeric enzymes with two active

sites. The subgroup Ia enzymes recognize different substrates by changes in relative

11

Chapter1

spatial disposition of large and small domains. A tabulated summary of the basis of

substrate recognition in the subgroups of the AAT enzyme family is given in Table 1.2.

1.4.2 Group II aminotransferases The A Ts in this subgroup include lysine f-aminotransferase (LA T), w-amino

acid aminotransferase (w-AAT) (Watanabe et al., 1989), ornithine aminotransferase

(OAT) (Shen et al., 1998) and "y'- aminobutyrate aminotransferase (GAB A) (Storici et al.,

1999). These enzymes exhibit broad substrate specificity and the active site lysine residue

occurs one position earlier in the polypeptide chain compared to other fold type I enzymes,

suggesting a deletion in ancestral group II aminotransferases (Kack et al., 1999). Enzymes

of the AA T family catalyze a reversible two-step reaction where one substrate-product

pair is usually a-ketoglutarate-L-glutamate while the other pair is dictated by the

enzyme's specificity for a particular substrate. The chemical changes occur at the eli atom

in L-glutamate; while changes in the case of the unique substrate may occur at the same

atom or in many cases like LAT, ornithine aminotransferase, or "y'-aminobutyrate

aminotransferase at the carbon atom attached to the distal amino group. The remaining

group II examples are dialkylglycine decarboxylase (DGD) (Toney et al., 1993),

glutamate 1-semialdehyde aminotransferase (GSA-AT) (Hennig et al., 1997) and 7, 8-

diaminopelargonic acid synthase (DapaAT) (Kack et al., 1999). In the case of DGD, the

common substrate is pyruvate and also the presence of three distinct sub-sites has been

reported. In GSA-AT, the mechanistic basis is different and there is no common substrate.

12

Chapter1

Q--cn-coo-

I NH3+

Amino acid 1 Amino acid 2

/NH/

H2C

(J-c--coo-II

0-

D--c-coo-

II o o

2-oxo acid of amino acid 1 PMP 2 oxo acid of amino acid 2

Scheme 1.2: The transamination reaction consists of essentially the two half-reactions. An aminotransferase has unique amino acid I as the substrate in the first half reaction and amino acid 2 (usually a common amino acid glutamate) in the second half-reaction. Aminotransferases thus recognize two different kinds of amino acids, the side chain of which are different in shape and properties. (Scheme is adopted from Hirotsu, et at., 2005).

1.4.3 Group III aminotransferases

In group III aminotransferases, structurally characterized examples are D-amino

acid aminotransferase (D-AAT) (Peisach et al., 1998) and branched-chain

aminotransferase (BCAT) (Goto et al., 2003). Unlike other ATs which belong to fold type

I, PLP dependent enzymes, BeATs and D-AATs belongs to fold type IV PLP dependent

enzymes. This is particularly interesting because active site is in part mirror image of the

L-amino acid aminotransferase (Sugio et al., 1995), and active site lysine is positioned on

the re face and the si face is solvent exposed, neatly accounting for its opposite stereo

specificity. The major structural difference between BCATs and D-AATs is the

hydrophobicity of the active site binding pockets i.e. the histidine and arginine residues are

13

Chapter1

located at the large pockets in D-AATs, whereas the tyrosine and phenylalanine residues

are at the small pocket in BCATs.

1.4.4 Group IV aminotransferases Phospho serine aminotransferase (PAT) is a structurally characterized example of a

group IV enzyme. In PAT, the substrates have side chains of equal lengths and they are

recognized by several active site arginines which are involved in making ion pairs and

hydrogen bonds with an Asp residue also (Hester et al., 1999). The role of a conserved

His41 within the group IV family in catalytic mechanism and substrate recognition has

also been suggested.

1.5 Dual substrate specificity in aminotransferases Molecular recognition is a phenomenon in which a chemical entity IS

specifically recognized from among a number of other molecules. This extraordinary

ability is achieved by specific interactions between the molecules. The molecular

recognition phenomenon is involved in enzyme catalysis, DNA replication and

trancription, immuneoresponse, signal transduction etc. An enzyme recognizes a

specific substrate from a number of other molecules in the cell, binds to it, catalyzes

and transforms it into a specific product.

PLP-dependent aminotransferases are key enzymes in amino acid metabolism

and reversibly catalyze the transamination reaction, which consists of essentially the

same two half reactions (Scheme 1.2) (Christen, 1985). An aminotransferase accepts

first amino acid which is specific for each enzyme in the first half reaction and the

second amino acid which is common, glutamate, in most aminotransferases in the

second half reaction. The amino group from first amino acid is transferred to the PLP

resulting in Pyridoxamine 5' -phosphate (PMP) and 2-oxo acid derivative of the first

amino acid. In the next reaction 2-oxo acid of second amino acid (a-ketoglutarate)

accepts the amino group of PMP to yield the common amino acid (glutamate) and

regenerates PLP. The dual substrate recognition of aminotransferases is essential for

amino acid metabolism. It is conceivable that this feat requires sophisticated design of

the active site compared to enzymes involved in single substrate recognition (Hirotsu

et al., 2005).

14

Chapter]

The basis of dual substrate specificity of some ATs has been elucidated by

recent structural information. Because the reaction requires two different substrates to

bind in succession to the same co-factor in the active site, these enzymes must be able

to accommodate both substrates while discriminating among all others. One possible

solution would be for the PLP itself to move between binding sites of the two different

substrates, but such a movement of the PLP has never been observed (Eliot et al.,

2004). An alternative is to take advantage of flexibility of side chains to position the

functional groups into exclusive binding sites.

The problem of dual substrate specificity is generally that of accommodating the

negatively charged ),-carboxylate of glutamate in a site that must also accept a neutral or

positively charged side chain. To achieve this, different groups of enzymes have adopted

different mechanisms. A tabulated summary of the basis of substrate recognition in the

subgroups of the AAT enzyme family is given in Table 1.2. Inter domain displacements

leading to 'closed' and 'open' forms have been observed in group I enzymes like AAT

between structures involving the liganded and unliganded forms. These changes have been

linked to modifying substrate specificities in the enzyme's half reactions. Binding of the

common C5 substrates induces the 'open' form of the enzyme while unique substrate

recognition requires a change to the 'closed' form (Islam et al., 2003 & Islam et al., 2005).

The conformational change is due to the small domain movement towards the large

domain to close the active site shielding the substrate from solvent region. Another

subclass of group I enzymes are those whose unique substrate mostly contain an aromatic

side chain. In this subclass smaller sub-domain movements accompanied by specific

recognition strategies have been suggested for individual enzymes. For example, aromatic

aminotransferase from P. horikoshii (Matsui et al., 2000) and E.coli, methionine

aminotransferase (Dolzan et al., 2004) utilize similar strategies involving large scale

rearrangements of the active site hydrogen bonding network to specifically distinguish

between substrates. On the other hand, histidinol phosphate aminotransferase from E.coli

(Haruyama et al., 2001) and T maritima (Fernandez et al., 2004) and glutamine

aminotransferase from T thermophilus (Goto et al., 2004) utilize small sub-domain

movements involving the N-terminal regions and associated closed-open conformational

changes for recognition of substrates. In the E. coli and T cruzi tyrosine

aminotransferases, the PLP binding mode is implicated in substrate recognition with the

phosphate group reportedly directing the incoming substrate.

15

Chapter1

Crystallographic and modeling studies on group II aminotransferases have given

insights into the different strategy employed by E. coli GABA (Liu et al., 2005) and

Human OAT (Markova et al., 2005), which react with both w- amino acid substrates and

the common substrate glutamate. GABA like other AATs and TATs bind the dicarboxylic

acid substrate via two conserved arginines. The carboxylate of w- amino acid occupies the

same position as the ),-carboxylate of glutamate, thereby taking advantage of similar

distance between amino and carboxylic group of unique substrate and amino and )'­

carboxylate of glutamate. Subsequently recent mutational study on human OAT

suggested, among other things, that conversion of the enzyme to its PMP form disrupts the

internal Glu-Arg interaction and enables the binding of a-ketoglutarate. It was also

suggested that the Glu-Arg interaction should be disengaged before glutamate can bind to

the unliganded form of that enzyme. The remaining group II examples are DGD (Toney et

al., 1993), GSA-AT (Hennig et al., 1997) and DapaAT (Kack et al., 1999) respectively. In

the case of DGD, the common substrate is pyruvate and also the presence of three distinct

sub sites has been reported. In GSA-AT, the mechanistic basis is different and there is no

common substrate. Specificity in the reaction is generated by a Glu-406 which is

positioned to repel a-carboxylic acids and a mobile loop that closes on the active site upon

substrate binding. In DapaAT, the substrates are S-adenosyl-L-methionine and 7-keto-8-

aminopelargonic acid. An Arg391 plays an important role in substrate binding in this

enzyme, but no overall inter-domain movement is observed in these structures as also in

other group II enzymes.

Group III enzymes have a completely different fold but convergent evolution has

apparently led to striking similarities in active site architectures; also, their modes of

distinguishing substrates appear quite different because domain movement is not observed

(Goto et al., 2003 & Peisach et al., 1998). In BCAT, the a-carboxylate of the substrate

binds to the hydroxyl group of a Tyr95 and also the main chain NH of Thr257 and Ala258

are involved. Glutamate recognition was suggested to be due to interactions with Arg97,

hydroxyl groups of Tyr129, Tyr31* and Vall 09*.

PAT is a structurally characterized example of a group IV enzyme. In PAT, the

substrates have side chains of equal lengths and they are recognized by several active site

arginines, which are involved in making ion pairs and hydrogen bonds with an aspartate

residue also (Hester et al., 1999). The role of a conserved His41 within the group IV

family in catalytic mechanism and substrate recognition has also been suggested.

16

Chapter1

1.6 Structures of group II aminotransferases Structurally characterized enzymes from this subgroup of ATs comprises lysine €­

aminotransferase (LAT) (the present work), w-amino acid aminotransferase (w-AAT),

ornithine aminotransferase (OAT), "(- aminobutyrate aminotransferase (GABA) ,

dialkylglycine decarboxylase (DGD), glutamate I-semialdehyde aminotransferase (GSA­

AT) and 7, 8-diaminopelargonic acid synthase (Dapa-AT). They invariably function as a

homodimer or higher order oligomer, with two active sites per dimer. Group II

aminotransferases are characterized by its low sequence similarity but the overall fold is

quite conserved.

1.6.1 ),-Aminobutyrate aminotransferase This enzyme operates by two half reactions. In the first half-reaction, GABA is

converted to succinic semialdehyde and its ,,(-amino group is transferred to the co-factor to

generate PMP. In the second half-reaction, a-ketoglutarate is converted to L-glutamate and

the PLP form of the enzyme is regenerated. The biologically active oligomer of E. coli

GABA-AT is an C4-tetramer with 426 residues per subunit. Each of the four monomers in

the asymmetric unit contains an N-terminal segment, a large PLP binding domain and a

small C-terminal domain (Fig 1.3A) (Liu et al., 2004 & Liu et at., 2005). The N-terminal

segment (residues 2 to 44) contains one tt-helix and a three-stranded antiparallellJ-sheet.

The large domain (residues 49 to 321) is comprised of a central seven-stranded IJ-sheet

surrounded by 11 a-helices. The C-terminal domain (residue from 323 to 426) is

composed of three a-helices and a four-stranded antiparallel IJ-sheet, and is at the comers

of the tetramer. The two PLP co-factors are located close to the subunit interface of the

dimer and close to each other (the PLP phosphorus atoms are 14.8 A apart). Both active

sites in the dimer are formed from residues contributed by both monomers. The C4' atom

of PLP originally forming an aldehyde with oxygen, connects covalently to the €-amino

group of Lys268 forming the internal aldimine linkage. In addition to the hydrogen bond

made with the aldimine nitrogen, the 3' phenolic oxygen of PLP makes a hydrogen bond

with the side chain amide nitrogen of Gln242, and interacts with a water molecule that is

additionally hydrogen bonded to the backbone nitrogen of Glu211 and the side chain

carboxylate of Glu206. The pyridine nitrogen atom of PLP makes a salt bridge (2.80A)

with Asp239. The phosphate group is held in the place by a total of nine hydrogen bonds

to the nonester oxygens (saturating its hydrogen bonding capacity), four of which are

17

Chapter]

donated by water molecules. The overall and active site structures of pig GABA-AT

(Storici et al., 1999) are quite similar to those of E. coli GABA-AT. The folds of the

monomers and the dimeric structures are highly conserved, while the higher order

quaternary structures vary.

1.6.1 Ornithine aminotransferase Ornithine aminotransferase (OAT), a pyridoxal-5'-phosphate dependent enzyme,

catalyses the transfer of the delta-amino group of L-ornithine to 2-oxoglutarate, producing

L-glutamate-,),-semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate,

and L-glutamate. The O!6-hexameric molecule is a trimer of intimate dimers (Shen et al.,

1998). The monomer fold is that of a typical representative of subgroup II

aminotransferases (Fig l.3B). The large domain contains the characteristic central seven­

stranded beta-sheet covered by eight helices in a typical et!(3 fold. The co-factor pyridoxal-

5'-phosphate is bound through a Schiff base to Lys292 located in the loop. The C-terminal

domain includes a four-stranded antiparallel beta-sheet in contact with the large domain

and three further helices at the far end of the subunit. The active sites of the dimer lie

about 25A apart at the subunit and domain interfaces. The conical entrances are on

opposite sides of the dimer. In the active site, Arg180, Glu235 and Arg413 are substrate­

binding residues. The (3-carboxylate of Asp263 makes a hydrogen-bonded ion pair with

the protonated N1. The crystal structure of the enzyme-inhibitor complex was solved at a

resolution of 1.95 A. No significant conformational changes in complex structure

compared with the native enzyme structure were observed (Storici et al., 1999). The

structures of human OAT bound to the inhibitors gabaculine and L-canaline were solved

to 2.3 A. Both inhibitors coordinate similarly in the active site, binding covalently to the

PLP co-factor and causing a 20° rotation in the co-factor tilt relative to the ligand-free

form (Shah et al., 1997).

18

Chapter]

Table1.2: Basis of substrate specificity in characterized enzymes of the aspartate aminotransferase family.

Group Enzyme

Ia AAT

ArAT

Ib AAT

ArAT

MetAT

HspAT

TAT

Source

E. coli (Okamoto et al., 1994), G. gallus (Malashkeivch et al.,1995), S. scrofa (Rhee et al., 1993) Paracoccus denitrificans (Okamoto et al., 1998)

Specific substrate

Aspartate

All aromatic amino acids

Thermus Aspartate thermophillus (Nakai et al., 1999)

Pyrococcus horikoshii (Matasui et al., 2000)

E. coli (Dolzan et al., 2004)

E. coli (Haruyama et al., 2001), Thermotoga maritime (Fernandez et al., 2004) Trypanosoma cruzi (Blankenfeldt et al., 1994), E.coli (Ko et al., 1999)

All aromatic amino acids

Methionine

Histidinol phosphate (Hsp)

Aromatic amino acids (Tyrosine preferred)

Substrate2

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

Pyruvate

Basis of substrate specificity

Recognizes different substrates by changes 111

relative spatial disposition of large and small domains.

Substrate recognition is through domain movement and rearrangement of hydrogen bond network. Grossly similar to AAT.

N terminal region of small domain approaches the active site to interact with substrate. Binding of acidic substrate occurs as in AA T of sub-group Ia above. Both acidic and aromatic substrates have been modeled to bind to the same active site pocket by large scale rearrangement of the hydrogen bond network including water mediated interactions Unknown, but it was hypothesized that it binds to substrates in a similar manner as Ar A T from P. horikoshii Open-closed conformational changes on binding of Hsp substrate mainly due to associated changes in the N-terrninal region.

Substrate specificity depends on the cofactor's (PLP) binding mode. P04-

2 group probably directs the incoming substrate.

19

GlnAT

KAT

II OAT

ACO-AT

GABA-AT

LAT

DGD

GSA-AT

DapaAT

III BCAT

Thermus Glutamine thermophilus HB8 (Goto et al., 2004) Human (Rossi et ai., L-kynurenine 2004)

Human (Markova et Ornithine al., 2005)

T thermophilus Acetyl ornithine

E. coli (Liu et al., y-aminobutyric acid 2004), Sus scrofa (Storici et al., 2004)

M. tuberculosis L-Iysine

Burkholderia capacia 2-aminoisobutyrate (Malashkevich et al., 1999)

Synechococcus Glutamate 1-(Henning et al., semialdehyde 1997)

E.coli (Eliot et al., 2002),

E. coli (Goto et al.,

S-adenosyl-L­methionine (SAM)

Hydrophobic amino

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

a-Ketoglutarate

Pyruvate

7-keto-8-aminopelargonic acid (KAPA)

a-Ketoglutarate

Chapter 1

Sub-domain movement observed. The sub-domain from 14-31 and 319-332 moves to close active site when it binds the unique substrate Conformation of Tyrl 01 changes during the catalytic cycle and plays an important role in substrate recognition. It was hypothesized that Glu235 switch plays an important role in substrate specificity. No domain movement observed. Structural details not published - but the glutamate and arginine residues are found to be conserved in the sequence No domain movement observed. It was hypothesized that an uncompensated positive charge near active site (Glu211-Arg398) is detrimental to catalysis. Present Work

Three distinct binding sites (sub-sites) are there. Transamination of Keto acids to L-amino acids occurs in sub-site B. The Glu-Arg pair is not conserved; also the common substrate is different. A Glu-406 positioned to repel a-carboxylic acids is suggested to be important for the enzyme's reaction specificity. Also the enzyme has a mobile loop (153-181) that closes on the active site upon substrate binding. No overall large domain movement. Structural studies indicate that an Arg391 is important for recognition of KAP A, but not required for binding SAM. No inter domain movement occurs upon substrate binding. The same hydrophobic pocket with relatively

20

2003), Human (Yennawar et al., 2001)

D-AAT Bacillus sphaericus (Peisach et al., 1998)

IV AGT S. cerevisae (Meyer et aI., 2005), Human (Zhang et aI., 2003)

PAT E.coli (Hester et al., 1999), Bacillus

'" alcalophilus \.

(Dubnovitsky et al., 2005)

~~tJr65"

1737;-Sf

acids

D-amino acids Pyruvate

Alanine Glyoxylate

L-phosphoserine a-Ketoglutarate

\H-\63q~

Chapter]

localized hydrophilic sites can accommodate the hydrophobic and acidic side chains of substrates at the same position. Molecular fold of group III enzymes are different. Substrates bind in opposite directions. No domain movement is observed. Tyr30, Arg98*, HislOO* are suggested to be determinants of the enzyme's stereo specificity. Reason for substrate specificity is not speculated upon

Particular arrangement of active site residues is unique. Topology, co-factor and substrate binding modes are different compared to AAT.

21

Chapter1

1.6.3 Dialkylglycine decarboxylase One of the two available decarboxylase tertiary structures is that of dialkylglycine

decarboxylase, an enzyme that is atypical of the group, because like the aminotransferases

it catalyses two half reactions. The first half reaction is a decarboxylation-dependent

transamination of dialkyl-glycine in which elimination of CO2 from CO! of the substrate is

followed by protonation at C4' of the coenzyme. This produces enzyme with cofactor in

the pyridoxamine form and the reaction is completed by a standard transamination half

reaction with pyruvate. The structure of DKB has same fold as observed in other member

of group II aminotransferases (Fig 1.3C) (Toney et al., 1995 & Malashkevich et al., 1999).

1.6.4 7, 8-Diaminopelargonic acid synthase DAPA synthase catalyses the conversion of 7-keto-8-diaminopelargonic acid

(KAP A) to 7, 8-diaminopelargonic acid (DAP A). The E. coli enzyme is a homodimer with

molecular weight of 94 kDa. It is unique in among aminotransferases in that it uses S­

adenosyl-L-methionine (SAM) as amino group donor (Kack et al., 1999). Each enzyme

subunit consists of two domains, a large domain (residues 50-329) containing a seven­

stranded predominantly parallel {J-sheet surrounded by a-helices, and a small domain

comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic

dyad in the crystals, form the homodimeric molecule which contains two equal active

sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit

and the large domain of the second subunit (Fig 1.3D). The cofactor is anchored to the

enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The

phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113

located at the N terminus of an a-helix. The pyridine nitrogen forms a hydrogen bond to

the side-chain of the invariant residue Asp245. Upon binding of SAM the substrate for the

first part of the catalytic cycle and the external aldimine is formed between the substrate

and cofactor through a transaldimation process.

1.6.5 Acetylornithine aminotransferase Acetylornithine aminotransferase (AcOAT) is one of the key enzymes involved in

arginine metabolism and catalyzes the conversion of N-acetylglutamate semialdehyde to

N-acetylornithine (AcOrn) in the presence of L-glutamate. Each chain of the Salmonella

typhimurium AcOAT (sAc OAT) is 405 residues long and folds into 15 a-helices and 1813-

22

Chapter1

strands. The monomer can be divided into three domains viz. a small N-terminal domain

(residues 1-60), a large PLP-binding domain (residues 61-303), and a C-terminal domain

(residues 304-405) (Fig 1.3E). The fold of sAc OAT is similar to those of other enzymes of

the Type I subgroup II family of PLP. The dimer interface is mainly formed by the large

PLP-binding domains of the two subunits (Rajaram et at., 2008).

1.6.6 Glutamate-l-semialdehyde amino mutase

GSA-AT is an a2-dimeric protein which converts GSA to 5-aminolevulinate by

using PLP as cofactor. However unlike other aminotransferases, this enzyme converts

substrate to product by an intramolecular exchange of amino and oxo functions and has no

requirement for a-carboxylic amino or oxo acids to complete the reaction. The GSA-AT

fold can be divided into three domains. The N-terminal domain consists of about 70

residues. It is the main cofactor binding domain and contains a central seven stranded {3-

sheet with six parallel strands. The C-terminal domain comprising residues 327-433 folds

into a three-stranded antiparallel {3-sheet. It is covered on the outer surface with four

helices including one at the C-terminus (Fig 1.3F) (Hennig et at., 1997).

23

vZ

(a)

(g) (v)

qpd"AHOI

dld

Chapter]

2VEF.pdb 2GSA.pdb

(E) (F)

Fig 1.3: Ca ribbon tracing of GABA-AT (A), OAT (B), DKB (C), DAPA-AT (D), AcOAT (E) and GSA-AT (F) molecules viewed down the molecular two 2-fold axis. One subunit is drawn in cyan while other is shown in blue. The coenzyme PLP is represented by stick and red in color.

25

Chapter1

1. 7 Lysine f-aminotransferase The metabolism of lysine has been extensively studied but the mechanism of the

enzymatic deamination of lysine has remained unsolved. Lysine E-aminotransferase (LAT)

[EC.2.6.1.36] which belongs to group II aminotransferase family (Mehta et al., 1993),

catalyses a reversible transamination reaction between the L-Iysine and a-keto acids which

is in this case a-ketoglutaric acid. Fold type-I enzymes, of which the most widely studied

member is Aspartate aminotransferase (AAT) , have been further divided into several

subgroups. While AA T itself is a subgroup I enzyme, LAT is a member of subgroup II of

fold type-I aminotransferases (Hayashi, 1995). It is generally understood that the overall

reaction in this enzyme family proceeds via a ping-pong bi-bi mechanism (Velick et al.,

1962).

Lysine E-aminotransferase (LAT) is involved in L-Iysine metabolism in a wide

range of living organisms such as Candida guilliermondii (Der Garabedian et al., 1989),

Rodotorula gutinis (Kinzel et al., 1983), Flavobacterium lutescens IF03084 (Soda et al.,

1968a), Candida utilis (Hammer et al., 1992) etc. In many actinomycetes it is involved in

the ~-lactam antibiotic biosynthesis and its activity constitutes to be the first step in the

pathway (Fujii et al., 2000). LAT activity is specific to ~-lactam antibiotic producers and

is considered to be the first step in the ~-lactam antibiotic biosynthetic pathway (Tobin et

al., 1991). The gene encoding LAT was shown to be located in the ~-lactam antibiotic

gene cluster in both Streptomyces clavuligerus (Khetan et al., 2000) and Nocardia

lactamdurans (Coque et al., 1991), whereas lat is absent from genome of most other

actinomycetes, confirming that this enzyme is specific for secondary metabolism.

Soda et aI., in 1968 first characterized lysine E-aminotransferase from

Flavobacterium lutescens and showed that this enzyme transfers E-amino group of L­

lysine to a-ketoglutarate and yields L-glutamate and a-aminoadipate-o-semialdehyde

which is immediately converted into the intramolecularly dehydrated form/).l-piperidine-

6-carboxylate. In continuation of this study (Yagi et al., 1991) showed significant

differences were found in the reaction rate, and the substrate and reaction specificities

between the two half reaction and overall reaction catalyzed by LAT. The half reactions

between an amino donor and the enzyme bound PLP, and also between an amino acceptor

and the bound PMP followed first order rate kinetics. Kern and coworker in 1980 (Kern et

al., 1980) reported that in Streptomyces lactamdurans which is also an actinomycetes

produce a-aminoadipate during the synthesis of cephamycin C antibiotic. Lysine E-

26

Chapter1

aminotransferase catalyzes the first reaction III the converSIOn of L-Iysine to ex-aminoadipate. Earlier studies on LAT from Flavobacterium fuscum demonstrated that it

specifically catalyses an overall reaction involving the transfer of the E-amino group of L­

lysine to ex-ketoglutarate to yield L-glutamate and ex-aminoadipate-o-semialdehyde. This

latter product is spontaneously converted to a dehydrated form ~ l-piperidine-6-carboxylic

acid (Soda et al., 1968b). Hammer et al., 1992 purified and biochemically characterized

L-Iysine ex-oxoglutarate from Candida utilis and showed that this enzyme is functional as

dimer with molecular weight of 40 kDa of one chain. The enzyme from Candida exhibits

absorption maxima at 280, 340 and 420 nm with 2 moles of PLP per mole of enzyme. The

aminotransferase from Candida has a maximum activity at pH 8.5 with ex-oxoglutarate as

best amino acceptor and L-Iysine as amino donar.

The two half reactions in LA T are in Scheme 1.3 as also the molecular structure of

the substrates and products. An intriguing aspect of the reaction is the structural basis for

specific transamination of the distal amino group of L-Iysine in the first step while the ex-amino group of L-glutamate is involved in the second step.

M. tuberculosis exhibits significant changes in gene expreSSIOn during the

latent/persistent stage of infection. Proteomic analysis of the nutrient starved/latent phase

bacteria suggested a decreased expression of proteins involved in energy metabolism, lipid

biosynthesis and cell division in addition to induction of stringent response and several

other genes that may playa role in long-term survival within the host (Betts et al., 2002).

In M tuberculosis H37Rv, the Rv3290c gene has been annotated as a putative LAT in

databases. This gene was also found to be over 40-fold up-regulated in nutrient starved

tuberculosis models (Betts et al., 2002). Another study involving adoptation of the

pathogen to stationary phase and non-replicating persistence has shown that LAT is also

up-regulated under these conditions (Voskuil, 2004). Its regulation level, on the other

hand, drops down in long term latency (Voskuil et al., 2004). These studies agree well

with a time-course study of the expression of LA T during solid phase growth of

Streptomyces clavuligerus also where it was suggested that high LA T expression was

observed in response to nutrient deprivation which subsequently decreased to below

detectable levels after sporulation (Khetan et al., 2000). All together these studies seem to

suggest an important role for LATin adaptation to long term persistence in M.

tuberculosis. Genes that are upstream of sigF are up-regulated under nutrient starvation

condition are therefore of interest as potential targets for novel antibacterial agents. Lat is

27

Chapter1

one of those genes which is a member of the aspartate aminotransferase (AA T) family of

pyridoxal 5' -phosphate (PLP) dependent enzymes. MtLA T subunit has a molecular weight

of ~50 kDa which requires PLP as the cofactor for its catalysis.

Reaction -1 0-

+

NH/

E-PLP L-Lysine

Reaction-2

o

+ 0-

E-PMP a -Ketoglutarate

0-o

NH/

+ O~ 0-

E-PMP a -Aminoadipate.o -semialdehyde

il-H20

UCOOH N

/::::,.1-Piperideine-6-carboxylic acid

~\ 0-/

NH3+

~ OP03- I ~ 0- + NH+

0-0

E-PLP Glutamate

Scheme 1.3: The two half reactions in the overall reaction of lysine f-aminotransferase. In the first half reaction, L-Iysine reacts with the pyridoxal form of the holoenzyme (E-PLP; the active site K300 binds the cofactor via a Schiff base) to yield the oxoacid product, a-aminoadipate­o-semialdehyde, and the pyridoxamine form of the enzyme (E-PMP; no covalent bond to K300). a-aminoadipate-o-semialdehyde subsequently dehydrated to form /),1-piperidine-6-carboxylate. The second half-reaction is proceeds in the reverse direction. The substrate a-ketoglutarate reacts with E-PMP reconstituting E-PLP and releasing L-glutamate as product.

28