Review

Protein engineering of formate dehydrogenase

Vladimir I. Tishkov a,*, Vladimir O. Popov b

a Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119992, Russiab A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia

Received 23 November 2005; received in revised form 3 February 2006; accepted 6 February 2006

Abstract

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in

dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their

application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at

improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in

Escherichia coli cells.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Formate dehydrogenase; Protein engineering; Pseudomonas sp.101; Candida boidinii; Stability; Mutagenesis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

2. Approaches applied to FDH engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

2.1. Structure analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

2.2. Amino acid sequences alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

2.3. Random mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

3. Catalytic mechanism studies and improvement of kinetic parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

3.1. Switch in substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

3.2. Enhancement of catalytic activity of FDH from C. boidinii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

4. Improvement of FDH operation stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

4.1. Improvement of chemical stability of FDHs from Pseudomonas sp.101 and M. vaccae N10 . . . . . . . . . . . . . . . . . . . . . . 99

4.2. Improvement of chemical stability of FDH from C. boidinii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

5. Improvement of FDH thermal stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

5.1. Comparison of FDHs thermostability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

5.2. Improvement of PseFDH thermal stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

5.3. Improvement of CboFDH thermal stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

6. Change of coenzyme specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

7. Expression of FDH genes in E. coli cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

8. Alternative enzymes for NAD(P)H regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

8.1. Glucose dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

8.2. Phosphite dehydrogenase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

www.elsevier.com/locate/geneanabioeng

Biomolecular Engineering 23 (2006) 89–110

* Corresponding author. Tel.: +7 495 939 3208; fax: +7 495 939 2742.

E-mail addresses: vit@enz.chem.msu.ru, vitishkov@gmail.com (V.I. Tishkov), vpopov@inbi.ras.ru (V.O. Popov).

1389-0344/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.bioeng.2006.02.003

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–11090

1. Introduction

NAD+-dependent formate dehydrogenase (EC 1.2.1.2,

FDH) catalyzes oxidation of formate ion into carbon dioxide

coupled to the reduction of NAD+ to NADH:

HCOO� þNADþ ! NADH þ CO2:

The enzyme was first discovered in pea seeds more than

60 years ago (see Mathews and Vennesland, 1950; Davidson,

1951 and references therein). The intense studies began in 70s

of the last century, mostly for formate dehydrogenase from

methylotrophic bacteria and yeast. The interest originated from

both practical application of FDH for the purposes of NADH

regeneration in the enzymatic processes of chiral synthesis with

NAD+-dependent dehydrogenases (Wichmann et al., 1981;

Hummel and Kula, 1989), and fundamental studies on the

dehydrogenase catalytic mechanism. FDH belongs to the

superfamily of D-specific dehydrogenases of 2-hydroxy acids

(Vinals et al., 1993). All enzymes of this family have similar

structure and almost identical set of catalytically essential

amino acid residues in the active center (Popov and Lamzin,

1994; Lamzin et al., 1995). The choice of FDH as a model

enzyme was based on the fact that the enzyme catalyzes the

simplest reaction among the other enzymes of the superfamily

devoid of any proton release or abstraction steps.

Last decade active sequencing of genomes resulted in the

discovery of FDH genes in various organisms including

pathogens such as Staphylococcus aureus (Baba et al., 2002),

Mycobacterium avium subsp. paratuberculosis str.k10 (Li

et al., 2005), different strains of Bordetella (Parkhill et al.,

2003) and Legionella (Chien et al., 2004; Cazalet et al., 2004),

Francisella tularensis subsp. tularensis SCHU S4 (Larsson

et al., 2005), Histoplasma capsulatum (Hwang et al., 2003),

Cryptococcus neoformans var. neoformans JEC21 (Loftus

et al., 2005), etc. It was shown that, under specific conditions,

FDH could play a key role in cell functioning. For instance,

FDH appears to be a stress protein in plants. The enzyme

localizes to mitochondria and its biosynthesis sharply increases

(up to 9% of total mitochondrial proteins) under stressful

conditions (Colas des Francs-Small et al., 1993). The analysis

of FDH isoforms ratio was used to identify diseased trees

(Weerasinghe et al., 1999). In the case of S. aureus, FDH is one

of three overexpressed proteins, when the bacterium grows at

biofilm conditions (Resch et al., 2005). Bacterial biofilm

infections are particularly problematic because sessile bacteria

can often withstand host immune responses and are generally

much more tolerant to antibiotics, biocides and hydrodynamic

shear forces than their planktonic counterparts. Expression of

FDH gene is also phase specific in fungal pathogens (Hwang

et al., 2003).

The number of papers on FDH grows year by year, and

the majority of the works describes FDH application for

cofactor regeneration in the processes of chiral synthesis with

NAD(P)+-dependent oxidoreductases. General scheme of

NAD(P)H regeneration for cofactor coupled enzymatic

synthesis of optically active compounds is presented in the

next scheme:

The main enzyme Ep (dehydrogenase, reductase, monooxy-

genase, etc.) catalyzes production of a chiral compound using

reduced cofactor, while the second enzyme ER (for example,

formate dehydrogenase) reduces oxidized coenzyme back to

NAD(P)H. In some cases, the same enzyme can catalyze both

reactions (Hummel and Kula, 1989).

Numerous studies demonstrated that FDH is one of the best

enzymes for the purposes of reduced cofactor regeneration

(Shaked and Whitesides, 1980; Kula and Wandrey, 1987;

Hummel and Kula, 1989; Liese and Villela, 1999; Burton,

2003; Liese, 2005; Wichmann and Vasic-Racki, 2005). The

reaction catalyzed by FDH fits all the criteria for NAD(P)H

regeneration.

(1) T

he reaction is irreversible under normal conditions. This

provides thermodynamic pressure to shift equilibrium of

the main reaction and results in a 99–100% yield of the final

product.

(2) F

ormate-ion is a cheap substrate, and the reaction product,

CO2, can be easily removed from the reaction mixture and

does not interfere with the purification of the final product.

(3) F

DH exhibits a wide pH-optimum of catalytic activity (6.0–

9.0) (Mesentsev et al., 1997).

(4) M

ethanol-utilizing yeast and bacteria provide a high scale

enzyme production with a comparatively low production

cost.

(5) B

acterial and yeast FDHs are sufficiently stable to be used

in flow-through reactors for a while.

All the above factors determined the use of yeast FDH

from Candida boidinii for the purpose of NADH regeneration

in the first commercial process of chiral synthesis of tert-L-

leucine with dehydrogenase realized by ‘‘Degussa’’ (Bom-

marius et al., 1995). The process is still the biggest one in

production volume among the others used to produce

optically active compounds with the help of dehydrogenases.

Under the leadership of Profs. M.-R. Kula and C. Wandrey,

the methods for cultivation of C. boidinii yeast and enzyme

purification at the level of millions of activity units were

developed (Weuster-Botz et al., 1994).

Unfortunately, native FDHs have some disadvantages. First,

their operational stability is rather low due to the presence of

active Cys residues. Chemical modification or oxidation of

these residues results in fast enzyme inactivation. Second, there

are no native FDHs of the discussed family, which use NADP+

as a cofactor, and third, the production cost of FDH from native

strains of methylotrophic bacteria or yeast was still high enough

to use the enzyme in development of novel commercial

processes of chiral synthesis. The review summarizes the

experiments on FDH protein engineering based on directed and

random mutagenesis which permitted to produce a new

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 91

generation of biocatalysts for NAD(P)H regeneration exhibit-

ing improved and novel kinetic properties, increased chemical

and thermal stability, and lower production costs. Since the key

experiments have been performed with FDH from methylo-

tropic yeast C. boidinii and bacterium Pseudomonas sp.101,

these enzymes will be in the focus of the review.

One can specify the following directions for FDH studies

requiring mutagenesis:

- c

atalytic mechanism studies and improvement of kinetic

properties;

- in

crease in chemical stability;

- in

crease in thermal stability;

- s

witch in coenzyme specificity;

- c

rystallization and refinement of X-ray structure;

- in

crease in the level and rate of FDH gene expression in

Escherichia coli.

The available information on FDH mutations is summarized

in Table 1. Unfortunately, all experiments cannot be covered

within a single review, therefore, we limit ourselves to

consideration of the most important mutations which were

critical for the production of new generation of recombinant

FDH biocatalysts for NAD(P)H regeneration.

2. Approaches applied to FDH engineering

2.1. Structure analysis

X-ray data analysis was used to select mutation positions for

FDH from Pseudomonas sp.101 and highly homologous

(different in only two aa residues) FDH from Mycobacterium

vaccae N10. High resolution structures are available from PDB

for apo-PseFDH (2NAC) and the ternary complex (PseFDH-

NAD+-azide) (2NAD) resolved in 1993 (Lamzin et al., 1994).

Recently, some other complexes of the enzyme have been

crystallized and respective structures solved (Table 2). The

analysis of PseFDH structures in the complex with formate,

ADP-ribose, NADH and (NADH + formate) shows their

intermediate character between 2NAC and 2NAD structures,

i.e. apo-enzyme transformation into a holo-enzyme. All the

complexes have been obtained with native enzyme purified

from Pseudomonas sp.101. The presence of seven additional

amino acid residues at the C-terminus of recombinant wt-

PseFDH (Tishkov et al., 1991) interferes with crystallization.

The deletion of these residues by means of mutagenesis

resulted in production of crystals of recombinant FDH. The

crystals of full size 400 aa polypeptide have been produced for

two mutant forms with improved thermal stability, PseFDH

GAV and PseFDH T7.

High homology scores for FDH from different sources

(Fig. 1) allowed high accuracy model structures to be obtained

for the enzymes from C. boiidinii (Felber, 2001; Slusarczyk

et al., 2000; Labrou et al., 2000; Labrou and Rigden, 2001),

Candida methylica (Karaguler et al., 2004) and Saccharomyces

cerevisiae (Serov et al., 2002). These structures were

successfully used to plan mutations aimed at improving

chemical stability (Felber, 2001; Slusarczyk et al., 2000) and

studying the catalytic mechanism (Labrou et al., 2000; Labrou

and Rigden, 2001) of CboFDH, and for the switch in coenzyme

specificity of FDH from S. cerevisiae (Serov et al., 2002).

Numerous attempts to get wild-type CboFDH crystals

suitable for the structure resolution failed. To get the required

quality of CboFDH crystals, an approach based on the

introduction of amino acid replacements in the regions of

highly disordered structure, has been applied (Schirwitz et al.,

2005). The prediction of these regions for new enzymes is

performed using special programs and the structure of a

homologous enzyme. In the case of CboFDH, this approach

was used to introduce the following mutations: Lys47Val,

Lys47Glu, Arg296Ala, Lys328Val and Lys338Ala. Replace-

ment Lys47Glu in CboFDH resulted in preparation of high-

quality enzyme crystals, which provided 1.9 A resolution of the

apo-enzyme structure (Schirwitz et al., 2005). Noteworthy,

Lys47 (Lys75 in PseFDH) is conserved through all 51 FDH

complete and partial sequences known up to date (Fig. 1).

2.2. Amino acid sequences alignment

The above approach is widely used for all enzymes. It is

commonly used in combination with other methods. For

instance, the alignment of FDH amino acid sequences from

different sources was used to localize non-conserved Ser

residues while improving PseFDH thermal stability with

hydrophobization of a-helices (Rojkova et al., 1999) and

optimization of polypeptide chain conformation (Serov et al.,

2005). The approach has been also used to select the type of the

introduced residue while increasing chemical stability of

PseFDH (Tishkov et al., 1993; Odintseva et al., 2002),

MycFDH (Yamamoto et al., 2005) and CboFDH (Slusarczyk

et al., 2000) (see below). The comparative analysis of FDH

amino acid sequences led us to decision to clone FDH from S.

cerevisiae (Serov et al., 2002; Serov, 2002). It was the first

enzyme with Lys and Val residues upstream catalytically

important Gln313 and His332 (numbered as in PseFDH),

respectively, whereas the majority of FDHs contain Pro

residues in these positions (Fig. 1).

Until recently, the effectiveness of this approach was limited

by the small number of the cloned FDH sequences. In last 5

years, direct cloning of FDH genes and genome sequencing of

different organisms resulted in a whole series of complete and

partial sequences of the enzyme. Right now, 52 complete (17

from bacteria, 15 from plant, 10 from yeast and 10 from fungi)

and more than 15 partial FDH sequences are known. Fig. 2

presents evolution tree for FDHs from different sources,

generated with the Clustal X 1.83 program. The analysis did not

include enzymes from M. vaccae N10 and C. methylica, since

they differ in two replacements only (differences lower than

1%) from PseFDH and CboFDH sequences, respectively. Fig. 2

demonstrates that bacterial and plant FDHs form very compact

groups, which are rather far from other FDHs. The biggest

variety in sequences is observed for yeast and fungal FDHs.

Nevertheless, FDH is an extremely conserved enzyme. Among

all enzymes from all sources, 60 aa residues are absolutely

V.I.

Tish

kov,

V.O

.P

op

ov

/Bio

mo

lecula

rE

ng

ineerin

g2

3(2

00

6)

89

–1

10

92

Table 1

Mutations performed to improve formate dehydrogenases propertiesa

Aim Mutation/source Result/conclusion Ref.

Probing of molecular mechanism

Increase of specific activity C23S/F285S CboFDH 1.7-fold increase of specific activity, values of Kformatem

and KNADþm increased from 6 mM and 45 mM to

14 mM and 74 mM, respectively

Felber (2001)

Role of loop between b8-sheet and aA-helix

in substrate specificity

Glu141Gln, Glu141Asn, ParFDH Mutations Glu141Gln and Glu141Asn induced 5.5-

and 4.3-fold increases in Kformatem values, 110- and

590-fold decreases in the kcat for reaction with formate

and 9.5- and 85-fold increases in catalytic efficiency in

reaction of glyoxylate reduction, respectively

Shinoda et al. (2005)

Increase of operational stability

Change of ‘‘essential’’ Cys, controlling PseFDH

operational stability. Cys255 is located above the

plane of adenine moiety of NAD and

occupies conservative position

Cys255Met, Cys255Ser, Cys255Ala, PseFDH Stable at least a month (200-fold increase in chemical stability) Tishkov et al. (1993),

Odintseva et al. (2002)Decreased thermostability. KNADþm increased seven-fold

for Met, three-fold for Ser and is the same as for WT

for Ala; formate binding is unchanged for Ala and Ser

and is three-fold decreased for Met

Change of surface Cys354 Cys354Arg, Cys354Ser, Cys354Ala, PseFDH Provided best thermal stability Odintseva et al. (2002)

Cys255Ala/Cys354Ala, PseFDH 1000-fold increased operational stability Odintseva et al. (2002)

Change of Cys145 near

catalytically important Asn146

Cys145Ser No changes in kinetic parameters and thermal stability Own data

Cys145Ala, PseFDH No changes in kinetic parameters and 10%

decrease of thermal stability

Cys255Ala/Cys145Ser,

Cys255Ala/Cys145Ala, PseFDH

No changes in kinetic parameters and increase

of chemical stability >1000-fold

Change of essential Cys in MycFDH (PseFDH and

MycFDH differ by only two residues in

positions 35 and 61)

Cys6Ser, Cys145/Ser, Cys255Ala/Ser/Val,

C146S/C256V, C6A/C146S/C256V, MycFDH

No data about kinetic properties and thermal stability.

Increase of chemical stability was estimated by

tolerance to inactivation by substrate ethyl

4-chloroacetoacetate and the yield of synthesis

of ethyl (S)-4-chloro-3-hydroxybutanoate

Yamamoto et al. (2005)

Change of all available cysteines in CboFDH Cys23(52)Ser, CboFDH No change of kinetic parameters, increased chemical stabiliy Slusarczyk et al. (2000),

Felber (2001)Cys262(288)Val, CboFDH No change of kinetic parameters, diminished chemical stabiliy

Cys23Ser/Cys262)Ala, CboFDH No change of kinetic parameters, substantially

decreased thermostability but operational stability

under biotransformation conditions increased an

order of magnitude

Increase of thermal stability

Optimization of electrostatic interactions

(effect of amino residues in positions 43 and 61

on thermal stability of bacterial FDH)

Glu61Gln, Glu61Pro PseFDH and MycFDH differ by only

two residues in positions 35 and 61

Galkin et al. (1995),

Fedorchuk et al. (2002)

Glu61Lys, MycFDH Four- to six-fold lower thermostability of

MycFDH is caused by electrostatic

repulsion between Asp43 and Glu61 residues

Lys61Arg, PseFDH Mutation changed temperature dependence

of thermal inactivation rate constant

Hydrophobization of a-helices Ser131Ala 1.20-fold increase of thermal stability compared to wt-PseFDH Rojkova et al. (1999)

Ser160Ala 1.24-fold increase of thermal stability compared to wt-PseFDH

Ser168Ala 1.40-fold decrease of thermal stability compared to wt-PseFDH

V.I.

Tish

kov,

V.O

.P

op

ov

/Bio

mo

lecula

rE

ng

ineerin

g2

3(2

00

6)

89

–1

10

93

Ser184Ala 1.13-fold increase of thermal stability compared to wt-PseFDH

Ser228Ala 1.2-fold increase of thermal stability compared wt-PseFDH

Ser(131,160)Ala 1.40-fold increase of thermal stability compared o wt-PseFDH

Ser(184,228)Ala 1.28-fold increase of thermal stability compared o wt-PseFDH

Ser(131,160,184,228)Ala 1.60-fold increase of thermal stability compared o wt-PseFDH

(mutant T4) PseFDH The same kinetic properties as for wt-PseFDH

Tyr62Phe No change of thermal stability compared to wt- eFDH Serov and

Tishkov (2002)Tyr165Phe, PseFDH 17.6-fold decrease of thermal stability compared o wt-PseFDH

Minimization of conformational

tensions in polypeptide chain

His263Gly 1.30-fold decrease of thermal stability compared o wt-PseFDH Serov et al. (2005)

Ala191Gly No significant effect on the stability

Asn234Gly No significant effect on the stability

Asn136Gly 1.20-fold increase of thermal stability compared o wt-PseFDH

Tyr144Gly 1.40-fold increase of thermal stability compared o wt-PseFDH

Tyr144Gly + T4, PseFDH 2.30-fold increase of thermal stability compared o wt-PseFDH

Improvement of thermal stability of

CboFDH by directed evolution

Cys23Ser CboFDH (SM CboFDH) Decrease of thermal stability 6.7-fold and Tm 58compared to wtCboFDH

Slusarczyk

et al. (2000)

Arg178Ser SM CboFDH Increase of thermal stability 3.1-fold and Tm 38 mpared to SM Slusarczyk

et al. (2003)

Arg178Gly, SM CboFDH Increase of thermal stability 2.2-fold and Tm 28 mpared to SM

Asp149Glu, Arg178Ser, SM CboFDH Increase of thermal stability 6.7-fold and Tm 58 mpared to SM,

mutation Asp149Glu provides increase of therm stability 2.15-fold

Glu151Asp, Arg178Ser, SM CboFDH Increase of thermal stability 27.6-fold and Tm 9 compared to SM,

mutation Glu151Asp provides increase of therm stability 9.0-fold

Glu151Asp, Arg178Ser,

Lys356Glu, SM CboFDH

Increase of thermal stability 18-fold and Tm 88 mpared to SM,

mutation Lys356Glu provides decrease of therm stability 1.5-fold

Glu151Asp, Arg178Ser,

Lys306Arg, Lys356Glu, SM CboFDH

Increase of thermal stability 18-fold and Tm 88 mpared to SM

Glu151Asp, Arg178Ser,

Lys306Arg, Thr315Asn, SM CboFDH

Increase of thermal stability 36-fold and Tm 108 ompared to

SM, mutations Lys306Arg and Thr315Asn prov e increase

of thermal stability 1.5-fold. This mutant is 5.7- ld more

stable than wt-CboFDH

Cys23Ser, Cys262A, CboFDH

(DM CboFDH)

Decrease of thermal stability 35-fold and Tm 10

compared to wtCboFDH

Slusarczyk

et al. (2000)

Lys306Arg, Thr315Asn, Lys356Glu,

DM CboFDH

Increase of thermal stability 3.8-fold and Tm 48 mpared to DM Slusarczyk

et al. (2003)

Glu18Asp, Lys35Arg, Arg187Ser,

DM CboFDH

Increase of thermal stability 3.8-fold and Tm 48 mpared

to DM, mutations Glu18Asp and Lys35Arg prov e increase

of thermal stability 1.1-fold

Slusarczyk

et al. (2003)

Glu18Asp, Lys35Arg, Glu151Asp,

Arg187Ser, Phe285Tyr, DM CboFDH

Increase of thermal stability 47-fold and Tm 118 ompared

to DM. This mutant is 1.4-fold more stable than t-CboFDH

Slusarczyk

et al. (2003)

Investigation of the role of conservative

prolines in thermal stability

Pro288(312)Thr, CboFDH Thermal inactivation rate increased 18-fold N. Labroub

Role of ‘‘charge-relay’’ system in thermal stability Gln287(313)Glu/His311(332)Gln,

CboFDH

Neutral mutation Stability increased 1.6-fold at 8C N. Labroub

Testing of role of Thr169 and Thr226 in

stability of C. methylica FDH

Thr169Val Decrease of kcat 4-fold compared to wt-CmeFDH Karaguler

et al. (2004)

Thr226Val No change of kinetic parameters compared to w CmeFDH

Thr169Val/Thr226Val, CmeFDH Decrease of enzyme stability by �4 kcal/mol du to remove

of hydrogen bond between this residues

to

t

t

t

Ps

t

t

t

t

t

co

co

co

al

8al

co

al

co

c

id

fo

8

co

co

id

c

w

55

t_

e

V.I.

Tish

kov,

V.O

.P

op

ov

/Bio

mo

lecula

rE

ng

ineerin

g2

3(2

00

6)

89

–1

10

94

Table 1 (Continued )

Aim Mutation/source Result/conclusion Ref.

Change of coenzyme specificity

Change of coenzyme specificity of FDH

from C. methylica from NAD+ to NADP+

Asp195(221)Ser Decrease in coenzyme preferencec for NAD+ from 2.5 � 105 to 410 Gul-Karaguler

et al. (2001)CmeFDH The mutant enzyme still retained specificity for NAD+

Change of coenzyme specificity of

CboFDH from NAD+ to NADP+

Asp195Ser Activity with NAD+ and NADP+ 1.5 and 0.083 U/mg, respectively Rozzell

et al. (2004)Asp195Ser/Tyr196His Activity with NAD+ and NADP+ 1.3 and 0.19 U/mg, respectively

Asp195Ser/Tyr196His/Lys356(379)Thr Activity with NAD+ and NADP+ 1.3 and 0.36 U/mg, respectively

CboFDH Final mutant is 276-fold more active with NADP+ compared to

wt-CboFDH. No data about Km for formate and coenzymes

Change of coenzyme specificity of

SceFDH from NAD+ to NADP+

Asp196(221)Ala/Tyr197Arg, SceFDH Shift in coenzyme preference for NAD+ from >3 � 109 to

0.43–0.67 resulted in NADP+-specific enzyme

Serov et al. (2002)

Change of coenzyme specificity of

PseFDH from NAD+ to NADP+

PseFDH T5M8 Shift in coenzyme preference for NAD+ from 2.4 � 103

to 0.29 resulted in NADP+-specific enzyme, KNADPþm is

constant in pH range 6.0–7.0. The mutant enzyme has

specific activity with NADP+ 2.5 U/mg

Serov et al. (2002)

Extending of pH-optimum of NADP+

binding for mutant PseFDH

PseFDH T5M9-10 KNADPþm is constant in pH range 6.0–9.0 InnoTech

MSU (2005)

Preparation of enzyme crystals for x-ray analysis

Preparation of mutant CboFDH producing

crystals suitable for X-ray analysis

Lys47(75)Glu CboFDH Determination of apo-CboFDH structure with resolution 1.9 A´

Shwirwitz

et al. (2004)

a Numbering of the residues refers to particular enzyme, in parenthesis—numbering for PseFDH.b Prof. N. Labrou, personal communication.c The value of coenzyme preference for NAD+ is expressed as ðkcat=KmÞNADþ=ðkcat=KmÞNADPþ

.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 95

Table 2

Study of formate dehydrogenase structures by X-ray analysis

Enzyme form Resolution (A) Remarks

apo-PseFDH 1.8 Crystals prepared from native enzyme from Pseudomonas sp.101. Structure 2NAC (Lamzin et al., 1994)

PseFDH + NAD+ + azide 2.0 Crystals prepared from native enzyme from Pseudomonas sp.101. Structure 2NAD (Lamzin et al., 1994)

PseFDH + NADH 2.1 Native enzyme. Conformation similar to apoPseFDH (2NAC) (Filippova et al., 2005)

PseFDH + formate 2.2 Native enzyme. Formate is bound with Arg201, which is a residue responsible for binding of

pyrophosphate moiety of coenzyme. Cys354 is oxidized (Filippova et al., 2006)

PseFDH + ADP-ribose 1.5 Native enzyme. Only part of ADP-ribose can be seen. Conformation similar to apoPseFDH

except movement of residues 121–123

PseFDH + NADH + formate 2.3 Native enzyme. NADH is not visible in active site. Cys354 is oxidized. Conformation is

transient between apo-PseFDH and ternary complex with NAD+ and azide (Filippova et al., 2006)

PseFDH GAV 2.0 Mutant recombinant full size enzyme. Conformation similar to apoPseFDH

PseFDH T7 2.0 Mutant recombinant full size enzyme. Conformation similar to apoPseFDH

apo-CboFDH 1.9 Mutant CboFDH Lys47Glu (Schirwitz et al., 2005)

apo-Moraxella FDH 2.4 Recombinant enzyme expressed in E. coli. Active site has more open conformation than in apo-PseFDH

apo-ArabidopsisFDH 2.0–2.2 Recombinant enzyme expressed in E. coli. Coenzyme binding domain is similar to one in apo-PseFDH

conserved, and within the individual groups, the homology

score is higher than 75%.

2.3. Random mutagenesis

The above approach was successfully used to improve

thermal stability (Slusarczyk et al., 2003), increase catalytic

activity (Felber, 2001; Slusarczyk et al., 2003) and switch the

coenzyme specificity (Rozzell et al., 2004) of CboFDH.

Mutations were introduced with error prone PCR. The analysis

of E. coli cell libraries with mutant CboFDH (up to 200,000

clones) (Felber, 2001; Slusarczyk et al., 2003) were performed

in two steps. Primary qualitative screening of clones obtained

after transformation was carried out directly on solid agar. This

step yields only those clones that produced active enzyme.

Clones were selected in accordance with the protocol used for

the enzyme activity staining in PAAG (Felber, 2001). The

produced NADH was oxidized by phenazine etho-sulfate, and

the reduced form of the latter reacted with nitrotetrazoleum

blue to generate insoluble colored product. The yield of active

clones was only 0.1%, i.e. only one mutation out of 1000 did not

result in enzyme inactivation. At the second step, the selected

clones were cultivated in 96-well microtiter plates. Then, the

cells were lysed and the homogeneous enzyme was produced

using affinity microchromatography in 96-well microtiter

plates. To screen for mutant CboFDH with enhanced thermal

stability, the enzyme preparations were incubated at 50–58 8Cfor 15 min, and the residual activity was determined. To screen

for the mutants with high specific activity, the activity of free

enzyme was measured in the presence of a fixed concentration

of an inhibitor, Procion MX-R, which bound the enzyme

equimolarly (Felber, 2001).

To get NADP+-dependent CboFDH (Rozzell et al., 2004),

amino acid residues were replaced using error prone PCR,

however, in this case, the yield of active clones was much

higher than 70%, compared to the previous experiments

(Felber, 2001). All clones from the library (up to 20,000) were

cultivated in 96-well microtiter plates, and then, the enzyme

was isolated and its activity was analyzed with NADP+ in 96- or

384-well microtitre plates.

In conclusion of this chapter, we note that the rational design

based on the analysis of the enzyme structure and amino acid

sequences alignment saves one a lot of time and reagents to

generate new mutants. However, the modern level of computer

calculations for the effect of the introduced mutations does not

yield all possible candidatures for mutations. This can be

demonstrated by the improvement of CboFDH specific activity

(see below). Therefore, the maximum effect can be achieved

by combination of both approaches.

3. Catalytic mechanism studies and improvement ofkinetic parameters

3.1. Switch in substrate specificity

The model for FDH catalytic mechanism was proposed

using the structures of the apo-enzyme and of enzyme-NAD+-

azide ternary complex (Lamzin et al., 1994). The detailed

analysis of the effects of amino acid replacements on the

enzyme catalytic mechanism can be found in the review

(Popov and Tishkov, 2003). In addition to the mutations

reviewed there the effects of Glu141Gln and Glu141Asn

mutations in FDH from Paracoccus sp.12-A (Shinoda et al.,

2005) are discussed. As mentioned above, FDH belongs to the

superfamily of D-specific dehydrogenases of 2-hydroxy acids

and structure of D-lactate dehydrogenase is very similar to one

for PseFDH. The Asn97Asp replacement in D-lactate

dehydrogenase from Lactobacillus pentosus has minimum

effect on protein overall folding and catalytic activity,

however, the Km value for lactate increases 70-fold. The

Asn97 residue is located in the loop, which covers the enzyme

active center from the solvent. This residue is highly

conserved for majority of D-specific dehydrogenases of 2-

hydroxy acids. In the case of FDH Glu141 occupies the

equivalent position in 52 out of 53 known sequences. The

exception is the FDH from barley, which like E. coli D-lactate

dehydrogenase, contains Arg residue in the same position.

The Glu141Gln and Glu141Asn replacements in ParFDH

resulted in an increase of Km value for formate 5.5- and 4.3-

fold, a decrease in kcat for the reaction with formate 110- and

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–11096

Fig. 1. The alignment of amino acid sequences of formate dehydrogenases from bacteria Pseudomonas sp.101 (PseFDH) (Tishkov et al., 1991), Thiobacillus

sp.KNK65MA (TbaFDH) (Nanba et al., 2003a), Sinorhizobium meliloti (SmeFDH) (Barnett et al., 2001), Bordetella bronchiseptica RB50 (BbrFDH) (Parkhill et al.,

2003), Legionella pneumophila (LegFDH) (Chien et al., 2004), uncultivated g-proteobacterium EBAC31A08 (UmgFDH) (Beja et al., 2000), Mycobacterium avium

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 97

Fig. 2. Evolution tree for formate dehydrogenase. Full names of organisms from top to bottom: Emericella nidulans (Aspergillus nidulans), Aspergillus fumigatus

Af293, Ajellomyces capsulatus, Mycosphaerella graminicola (Septoria tritici), Gibberella zeae PH-1, Magnaporthe grisea, Botrytis cinerea, Neurospora crassa,

Saccharomyces cerevisiae, Candida albicans SC5314, Yarrowia lipolytica CLIB99, Pichia angusta (Hansenula polymorpha), Pichia pastoris, Candida boidinii,

Cryptococcus neoformans var. neoformans JEC21 (Filobasidiella neoformans), Ustilago maydis 521, soya G. max izoenzyme 2 and 1, Zea mays, rice Oryza sativa,

barley Hordeum vulgare, apple tree Malus � domestica, English oak Quercus robur, tomato Lycopersicon esculentum, potato Solanum tuberosum, Arabidopsis

thaliana, Streptomyces avermitilis, Mycobacterium avium subsp. paratuberculosis str.k10, Burkholderia sp.383, Bordetella bronchiseptica RB50 (Alcaligenes

bronchisepticus), Bordetella parapertussis strain 12822, Bordetella pertussis strain Tohama I, uncultivated marine g-proteobacterium EBAC31A08, uncultured

marine a-proteobacterium HOT2C01, Legionella pneumophila subsp. pneumophila str. Philadelphia 1, Sinorhizobium meliloti, Hyphomicrobium strain JT-17 (FERM

P-16973), Paracoccus sp.12-A, Moraxella sp., Ancylobacter aquaticus, Thiobacillus sp.KNK65MA and Pseudomonas sp.101.

590-fold, and an increase in catalytic efficiency in the

glyoxylate reduction 9.5- and 85-fold, respectively (Shinoda

et al., 2005). These results demonstrate the possibility to

change substrate specificity of the enzymes of superfamily of

D-specific dehydrogenases of 2-hydroxy acids.

3.2. Enhancement of catalytic activity of FDH from

C. boidinii

One of FDH disadvantages is its low catalytic activity.

The highest specific activity was reported for bacterial

subsp. paratuberculosis str.k10 (MavFDH) (Li et al., 2005), Streptomyces avermitilis

et al., 2000), potato Solanum tuberosum (PotFDH) (Colas des Francs-Small et al., 1

barley Hordeum vulgare (BarFDH, EMBL Accession D88272) and soya G. max (Soy

2000; Labrou et al., 2000) and Pichia angusta (HanFDH, former Hansenula polymorp

2004), Candida albicans (CabFDH) (Jones et al., 2004), S. cerevisiae (SceFDH, E

Aspergillus nidulans (AspFDH) (Saleeba et al., 1992), Magnaporthe grisea (MagFDH

XM_386303), Cryptococcus neoformans (CryFDH) (Loftus et al., 2005) and Ustilag

residues are shown in white letters on black background, conservative residues in

enzymes. At 30 8C, the specific activity of PseFDH is ca.

10 U per mg of protein. Enzymes from other sources are less

active than bacterial FDHs (Tishkov and Popov, 2004). The

activity of CboFDH is ca. 6.1–6.3 U/mg (30 8C) (Slusarczyk

et al., 2000; Labrou et al., 2000; Felber, 2001). However, due to

the difference in the molecular mass of these enzymes (44,000

and 40,370 Da for bacterial and yeast enzymes, respectively),

the values of kcat differ by two-fold, 7.3 and 3.7 s�1 for PseFDH

and CboFDH, respectively. The CboFDH activity was

increased up to 9.1 U/mg with random mutagenesis (Slusarc-

zyk et al., 2003). Out of 200,000 clones generated by random

(SavFDH) (Omura et al., 2001), plants Arabidopsis thaliana (AraFDH) (Olson

993), English oak Quercus robur (OakFDH, GeneBank Accession AJ577266),

FDH1); yeasts Candia boidinii (CboFDH) (Sakai et al., 1997; Slusarczyk et al.,

ha, EMBL P33677), Yarrowia lipolytica strain CLIB99 (YarFDH) (Dujon et al.,

MBL Z75296), fungi Ajellomyces capsulatus (AjeFDH) (Hwang et al., 2003),

, GeneBank Accession AY850352), Gibberella zeae PH-1 (CzeFDH, GenBank

o maydis (UstFDH, GeneBank Accession XM_402785). Catalytically important

bold. Residues subjected to mutagenesis marked by grey background.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–11098

Fig. 3. Position of Phe311PseFDH (Phe285CboFDH) (marked by pink color)

in ternary complex (PseFDH-NAD+-azide) (structure 2NAD). NAD+ and azide

are marked by dark blue and grey blue colors, respectively. Picture was created

using WebLab ViewerPro 3.7 software (Molecular Simulations Inc.).

mutagenesis, 1500 clones expressing the active enzyme have

been selected. Among the latter, four clones have been

identified with the enzyme specific activity higher than that

of the wild-type CboFDH. Sequencing showed that all four

clones had the same replacement, Phe285Ser. The authors

produced also a mutant CboFDH Phe285Tyr. The mutation

did not affect the enzyme activity, but increased its thermal

stability (see below).

The Phe residue in this position (Phe311 in PseFDH) is

highly conserved for bacterial FDHs: it is present in 15 from

Table 3

Location of cysteine residues in formate dehydrogenases

Position Bacteria Plants Yeasts Fungi Total Alternative resid

3 1 (17)a 0 (13) 0 (9) 0 (9) 1 (48) Val(6,0,6,6), Ile(

5 15 (17) 0 (13) 0 (9) 0 (9) 14 (48) Ala(1,0,0,4), Me

52 8 (17) 14 (15) 7 (9) 0 (10) 28 (51) Ser(9,1,0,0), Thr

74 0 (17) 0 (15) 0 (9) 1 (10) 1 (51) Ala(2,1,0,1), Ile(

81 3 (17) 6 (15) 0 (9) 0 (10) 9 (51) Ser(14,9,9,10)116 0 (17) 0 (15) 0 (10) 1 (10) 1 (52) Leu(14,16,8,9), I

117 0 (17) 1 (15) 7 (10) 2 (10) 9 (52) Ala(16,0,0,8), Le

140 1 (17) 1 (17) 0 (10) 0 (10) 2 (54) Ala(13,15,1,9), V

145 10 (17) 0 (17) 0 (10) 2 (10) 12 (54) Ser(7,17,10,8)171 3 (17) 0 (16) 0 (10) 0 (10) 3 (53) Trp(9,0,0,0), Gln

182 13 (17) 0 (16) 0 (10) 0 (10) 12 (53) Ala(3,0,2,4), Asp

196 1 (17) 0 (16) 0 (10) 0 (10) 1 (53) Thr(10,16,10,10)

199 0 (17) 0 (16) 0 (10) 1 (10) 1 (53) Gly(1,1,1,0), Ala

215 0 (17) 13 (15) 0 (8) 11 (11) 23 (51) Val(13,0,0,0), Pr

235 0 (17) 1 (14) 1 (10) 8 (11) 9 (52) Leu(12,0,1,0), A

248 13 (16) 16 (16) 1 (9) 9 (11) 39 (52) Ala(0,0,6,0), Val

255 11 (16) 0 (16) 6 (9) 11 (11) 27 (52) Ala(1,0,3,0), Ser

273 0 (16) 3 (16) 0 (9) 0 (11) 3 (52) Met(10,9,6,11), P

288 12 (16) 0 (16) 7 (9) 1 (11) 20 (52) Val(2,13,0,10), A

345 1 (16) 0 (15) 0 (9) 0 (11) 1 (51) Ala(15,14,9,11),

354 15 (16) 0 (15) 0 (9) 0 (11) 14 (51) Arg(0,15,0,1), Se

368 0 (16) 0 (13) 1 (8) 0 (11) 1 (48) Val(16,13,3,11),

Bold numbers show positions with high occurence of Cys and unique positions ofa 14 (16) means that 14 enzymes of 16 have Cys residue in this position.b Values in parentheses show number of alternative residue in bacteria, plant, ye

16 bacterial FDHs and only in FDH from Streptomyces

avermitilis this residue is substituted by homologous Tyr

(Fig. 1). Among 16 plant FDHs, there are 6 Phe, 4 Tyr, 5 Asn

and 1 Asp (Arabidopsis thaliana) residues in this position. In

20 sequences of yeast and fungal FDHs, Phe residue is found

12 times, Asp 5 times, Pro twice and Tyr once. The Phe285

(311 PseFDH) residue in FDH is in �2 position with respect to

the catalytically important Gln287 (313 PseFDH) residue,

which is located at the entrance to the active center of the

enzyme at the site of substrate-binding channel (Fig. 3).

Increase in CboFDH activity up to 9.1 U/mg resulting from

the Phe285Ser replacement did not effect the thermal stability

of the enzyme, but slightly worsened the Km values both for

coenzyme and formate. The Kformatem value grew from 6 to

14 mM, while KNADþm increased from 45 to 73 mM (Felber,

2001). We note that kcat value for the mutant CboFDH

Phe285Tyr, 6.1 s�1, is still lower than that for PseFDH, 7.3 s�1.

4. Improvement of FDH operation stability

The main reason for FDH inactivation at elevated tempera-

tures (up to 40–45 8C) is the oxidation of SH-groups of cysteine

residues. Chemical modification of Cys residues may occur due

to the impurities present in substrates or directly by substrates

containing active groups: for instance, ethyl 4-chloro-acetoactate

(ECAA) is used as a substrate for the synthesis of (S)-ethyl 4-

chloro-3-hydroxybutanoate ((S)ECHB), the key intermediate in

LipitorTM synthesis (Rozzell et al., 2004; Yamamoto et al., 2005).

Table 3 presents the data on the occurrence of Cys residues

in FDHs from various sources. As can be seen, bacterial FDHs

exhibit the highest content of Cys residues compared to the

uesb

9,13,3,1)

t(1,0,2,0), Gly(0,13,0,0), Leu(0,0,7,3)

(0,0,2,8), Ala(0,0,0,1)

0,0,1,0), Asp(0,10,3,0), Asn(1,0,0,0), Pro(0,3,0,0), Ser(14,1,4,8), Thr(0,0,1,0)

le(0,0,1,0), Ser(0,0,1,0), Met(3,0,0,0)

u(0,14, 2,0), Ile(1,0,0,0), Leu(0,0,1,0)

al(2,0,0,0), Met(1,0,0,0), Ser(0,1,0,0), Leu(0,0,6,0), Thr(0,0,3,0), Tyr(0,0,0,1)

(1,16,10,9), Ile(4,0,0,0), Met(0,0,0,1)

(1,0,0), Ile(0,13,3,1), Val(0,2,5,5), Met(0,1,0,0)

, Val(4,0,0,0), Ser(1,0,0,0), Gly(1,0,0,0)

(16,15,9,0), Val(0, ,0,8), Ser(0,0,0,1)

o(0,0,8,0,0), Met(2,0,0,0), Thr(1,0,0,0), Leu(1,1,0,0), Trp(0,1,0,0)

la(3,13,3,3), Val(2,0,4,0), Ile(0,0,1,0)

(3,0,0,0), Ser(0,0,2,0), Leu(0,0,0,1), Trp(0,0,0,1)

(1,0,3,0), Thr(1,13,3,0), Met(0,2,3,0), Val (1,0,0,0), Ile (1,0,0,0)

he(6,0,3,0), Leu(0,5,0,0)

la(1,3,0,0), Thr(1,0,2,0)

Thr(0,1,0,0),

r(0,0,6,10), Glu(0,0,2,0), Asp(1,0,0,0), Asn (0,0,01), (Val(0,0,1,0)

Leu(0,0,3,0), Ile(0,0,1,0)

Ser.

asts and fungi, respectively.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 99

enzymes from other sources. There are 14 positions in amino

acid sequences of bacterial FDHs where Cys residues can be

found; the probability of Cys occurrence in 7 positions is as

high as 70%. In plant enzyme Cys is found in eight positions,

and in three particular positions 52, 215 and 248 (numbered in

accordance with PseFDH sequence), the probability of their

occurrence is 93, 87 and 100%, respectively. In sequences of

yeast and fungal FDHs, Cys residues are found in 7 and 11

positions, respectively, among which only four are highly

conserved (Table 3).

Bacterial FDHs show the highest variability of Cys residues

content. The enzyme from Legionella pneumophila has the

highest number of Cys residue among all FDHs, nine per

subunit, and FDH from S. avermitilis has the lowest content,

one residue per subunit. Among 17 known bacterial FDH

sequences, 10 contain from six to eight Cys residues per

subunit. In plant and yeast FDHs, the average content of Cys

residues is from two to five per subunit.

As one can judge from Table 3, no correlation can be found

between Cys occurrence and FDH source. Only in one position

(248 in PseFDH) Cys residue is present in bacteria, plants, yeast

and fungi. There is also no specific preference for the residue

type for the positions that can be occupied by Cys, except

positions 81 and 145, which alternatively show Ser only (Fig. 1,

Table 3).

Cys residues show different activity and accessibility for

the solvent. Using apo-PseFDH structure (PDB2NAC) as

basic, one can mark out three groups of Cys residues. Most

solvent accessible are Cys81, 171, 255 and 354; much lesser

accessible are Cys residues in positions 52, 140, 145, 196,

248, 288 and 345. All others are located deep inside the

protein globule. The most critical for the enzyme activity are

Cys145 and Cys255. Cys145 is adjacent to Asn146, which

participates in formate binding in the enzyme active center,

and Cys255 is located in the coenzyme-binding domain and

contacts with the adenine moiety of NAD+ (Lamzin et al.,

1994).

4.1. Improvement of chemical stability of FDHs from

Pseudomonas sp.101 and M. vaccae N10

Each PseFDH subunit has seven Cys residues in positions 5,

145, 182, 248, 255, 284 and 354 (Fig. 1, Table 3). Chemical

modification experiments performed with PseFDH in the end

of 70s and beginning of 80s of the last century demonstrated

that modification of a single Cys residue per subunit was

sufficient to inactivate the enzyme. Amino acid sequencing

proved this residue to be Cys255 (Popov et al., 1990). The

PseFDH Cys255Ser and Cys255Met mutants produced in 1993

were absolutely stable toward Hg2+ ion inactivation and

showed a 100-fold decrease in the rate of inactivation with

DTNB (Tishkov et al., 1993). However, the mutants produced

showed inferior Km for substrates compared to wt-PseFDH,

and thermal inactivation rate increased four- to eight-fold

(Tishkov et al., 1993). The PseFDH Cys255Ala mutant

produced later had the same kinetic parameters as the wild-

type enzyme, but its thermal stability dropped four-fold

(Odintseva et al., 2002). Some native bacterial FDHs contain

Ala (Sinorhizobium meliloti) (Barnett et al., 2001), Val

(Thiobacillus sp.KNK65MA) (Nanba et al., 2003a), Ser (M.

avium subsp. paratuberculosis str.k10) (Li et al., 2005) or Thr

(S. avermitilis) (Omura et al., 2001) in 255 position, instead of

Cys (Fig. 1). It was found that FDH from Thiobacillus

sp.KNK65MA exhibited higher chemical stability against

inactivation with a-haloketones compared to the enzymes

from Ancylobacter aquaticus and C. boidinii (Nanba et al.,

2003a).

The chemical modification of PseFDH Cys255Ser and

Cys255Met mutants with DTNB showed the importance of an

additional Cys residue for the catalytic activity of PseFDH,

however, this second Cys was less reactive than Cys255

(Tishkov et al., 1993). A decrease in the inactivation rate for

the PseFDH Cys255Ser mutant under the action of DTNB in

the presence of formate-ion pointed to the residue localization

in the substrate-binding domain of the active center. This

second residue appeared to be Cys145, adjacent to Asn146

necessary for formate binding (Tishkov et al., 1991). Table 3

shows that all plant, yeast, fungal and six of bacterial FDHs

have Ser residue in this position. The Cys145Ser replacement

in PseFDH had no effect on kinetic parameters and thermal

stability, while the double mutant PseFDH Cys145Ser/

Cys255Ala exhibited at least a 1000-fold increase in chemical

stability compared to the wild-type enzyme. Single replace-

ment Cys145Ala slightly (10%) increased the rate of thermal

inactivation.

The analysis of apo- and holo-PseFDH structures demon-

strates the Cys354 accessibility for the solvent. In plant FDHs,

this position is occupied by Arg, and in yeast and fungi by Ser

(Table 3). The study of PseFDH mutant forms, Cys354Ala,

Cys354Ser and Cys354Arg shows that these replacements

increase Km for formate two- for four-fold, and decrease

thermal stability 2.5-, 3- and 10-fold, respectively (Odintseva

et al., 2002). X-ray analysis of (PseFDH + NADH + formate)

complex (two molecules per elementary crystallographic cell)

shows oxidized forms of sulfur in Cys residues: SO in one

subunit and SO3� in three others (Filippova et al., 2006). This

observation proves that Cys354 is not essential for chemical

stability and explains the appearance of different PseFDH

isoforms upon storage, due to multiple oxidation forms of

sulfur in this residue.

Improvement of chemical stability with directed mutagen-

esis was achieved for FDH from M. vaccae N10 as well

(Yamamoto et al., 2005). As we mentioned before, this enzyme

differs from PseFDH in two amino acid residues (Galkin et al.,

1995). In addition to Cys255Ala and Cys255Ser mutations, by

analogy with TbaFDH, the Cys255Val replacement was made

(Yamamoto et al., 2005). As for Cys145, all three mutations

were made, i.e. Cys145Ser, Cys145Ala and Cys145Val. In

addition, Cys5 was replaced for Ala, Val and Ser to generate

single, double and triple mutants. Unfortunately, the authors did

not analyze the properties of each mutant in detail. It was shown

that the introduced mutations resulted in a drop of enzyme

activity in cell-free extracts from 2- to 16-fold, and that the

activity of a triple mutant, Cys(5, 255, 354)Ser was only 0.011

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110100

compared to 3.24 U/mg for wt-MycFDH (Yamamoto et al.,

2005). The effectiveness of either mutation was evaluated by

the yield of the final product in the synthesis of (S)ECHB from

ECAA, and by the stability against 20 mM ECAA induced

inactivation at 25 8C. Analyzing the results of the work

(Yamamoto et al., 2005), one can conclude that Cys5 is not

essential for chemical stability of FDH. For instance, (S)ECHB

yield with MycFDH C5A/C145S/C255V triple mutant and

C145S/C255V double mutant was 32.2 and 31.0 g l�1,

respectively. The value of residual activity for the triple and

double mutants after 20 min incubation in the presence of

20 mM ECAA was 108 and 104%, respectively. These values

allow us to conclude that there is no recorded change within the

experimental error. In addition, for Cys145Ser/Cys255Val

double mutant, the activation effect in 5% ethyl acetate was

187% compared to 137% for C5A/C145S/C255V triple

mutant. The biggest activation was observed for C5A/

C145A/C255V triple mutant (219%), however, in this case,

the yield of the final product (S)ECHB was almost 20% lower

than for that of C5A/C145S/C255V mutant MycFDH.

Activation affect and increase of affinity for formate in

water-organic solvents were also shown for PseFDH (Dem-

chenko et al., 1990).

4.2. Improvement of chemical stability of FDH from

C. boidinii

CboFDH contains two Cys residues per subunit, Cys23 and

Cys262 (Ser52 and Cys288 in PseFDH, respectively) (Fig. 1).

Single Cys23Ser and Cys262Val, and double mutants

Cys23Ser/Cys262Val and Cys23Ser/Cys262Ala have been

produced (Slusarczyk et al., 2000; Felber, 2001). Cys23 plays

a more important role in chemical stability of CboFDH (Felber,

2001). For instance, in the presence of 150 mM hydrogen

peroxide, half-life periods (t1/2) for wt-CboFDH and its

Cys23Ser and Cys262Val mutants were 3.3, 7.3 and 2.4 min,

respectively, and in the presence of 50 mM CuSO4, t1/2 values

were equal to 38, 657 and 20 min, respectively (Felber, 2001).

These data are in good agreement with the results of computer

modeling of CboFDH structure (Slusarczyk et al., 2000; Felber,

2001). In accordance with the model, Cys23 is more solvent

accessible than Cys262. The most visual effect of CboFDH

chemical stabilization is observed at conditions for tert-L-

leucince production (40 8C and pH 8.2). Under these

conditions, the half-life time for Cys23Ser and Cys23Ser/

Cys262Ala CboFDH mutants increased more than five-fold

compared to the recombinant wt-enzyme (Slusarczyk et al.,

2000; Felber, 2001).

Values of Km for NAD+ and formate for single and double

mutants were unchanged compared to wt-CboFDH, however,

the specific activity decreased from 6.3 to 4.9–5.5 (Slusarczyk

et al., 2000). In addition, the introduced mutations resulted in

significant decrease in thermal stability of CboFDH. If single

Cys255Ala, Cys354Ala and Cys354Ser, and double

Cys255Ala/Cys354Ser mutations in PseFDH resulted in a 4-

, 2.5-, 3.0- and 10-fold increase in the rate of thermal

inactivation compared to the wild-type enzyme, respectively,

for single Cys23Ser, Cys262Val and double Cys23Ser/

Cys262Val 4 Cys23Ser/Cys262Ala of CboFDH, the rate of

thermal inactivation increased 6.7, 21.6, 93.7 and 35.1 times,

respectively, compared to the wild-type enzyme (Felber,

2001). Triple C145A/C255A/C354S PseFDH mutant exhib-

ited comparable thermal stability at 58 8C, and surpassed wt-

CboFDH at lower temperatures.

Thus, mutagenesis of Cys residues in FDH molecule results

in significant improvement of chemical stability coupled to

the decrease in thermal stability. To compensate the latter

effect, additional studies were needed to improve the enzyme

thermal stability.

5. Improvement of FDH thermal stability

5.1. Comparison of FDHs thermostability

There are many approaches in the literature to quantitatively

characterize enzyme thermal stability. In the case of FDHs,

many authors used the residual enzyme activity upon

incubation at a fixed temperature for a fixed time interval

(15–30 <4>) (Galkin et al., 1995; Shinoda et al., 2002; Nanba

et al., 2003a,b), or introduced the value of Tm, the temperature

which provides with 50% inactivation in 20 min (Slusarczyk

et al., 2000, 2003; Felber, 2001). The disadvantage of the first

approach is the difference in thermal inactivation mechanisms

for the enzymes from different sources, and inactivation

kinetics may be rather complicated. Therefore, the choice of

different time intervals could give opposite results. Moreover,

the mechanism of enzyme inactivation may change at elevated

temperatures. For instance, FDH from S. cerevisiae inactivates

reversibly at temperatures below 42 8C, while at elevated

temperatures, its inactivation mechanism includes both

reversible and irreversible steps (Serov, 2002).

Complex inactivation mechanism may cause serious differ-

ence in Tm-profiles for the same mutant series when different

time intervals are used. Moreover, Tm values give no clue to

quantitatively estimate enzyme thermal stability at temperatures

other than Tm. The most rational approach to characterize

enzyme thermal stability is to monitor the enzyme inactivation

kinetics at different temperatures, or to use differential scanning

calorimetry (DSC). The former approach gives quantitative

characteristics of enzyme stability at different temperatures.

The second method, DSC, allows the determination of the heat

of transfer between native and denatured states of the protein

globule.

Thermal stability of wt-CboFDH and its mutants was studied

in Slusarczyk et al. (2000, 2003) and Felber (2001). Quantita-

tive effects were presented as Tm and half-life period at 50 8C.

In this laboratory, the inactivation kinetics of wild-type and

mutant FDHs from bacteria Pseudomonas sp.101 (Rojkova et al.,

1999; Fedorchuk et al., 2002), M. vaccae N10 (Fedorchuk et al.,

2002) and Moraxella sp. as well as from yeast C. boidinii

(Sadykhov et al., 2006) and S. cerevisiae (Serov, 2002) and plants

A. thaliana and siya Glycine max have been studied (Sadykhov

et al., 2006). It was found that thermal inactivation of all FDHs

except that of S. cerevisiae is irreversible and follows kinetics

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 101

Fig. 4. Differential scanning calorimetry of wt-CboFDH, wt-PseFDH, mutant

PseFDH T7 with increased thermal stability and mutant PseFDH GAV with

increased chemical and thermal stability. Normalized melting curves. Protein

concentration 1 mg/ml, 0.1 M phosphate buffer, pH 7.0, heating rate 0.1 grad

per min.

Table 4

Tm values and first order inactivation rate constants of formate dehydrogenases at 55 8C

Type of enzyme kin (�106 s�1) kin=kPseFDHin

Tm (8C) Tm � TPseFDHm (grad)

wt-FDH Thiobacillus sp.KNK65MAa (Nanba et al., 2003a) 1330 274 52.5 �10.5

wt-FDH Ancylobacter aquaticusa (Nanba et al., 2003b) 996 205 53 �10

wt-FDH Paracoccus sp.12-Aa (Shinoda et al., 2002) 385 79 56 �7

wt-FDH Candida boidiniib 183 38 56.8 �6.2

Mutant Candida boidinii FDH C23Sb (Slusarczyk et al., 2000; Felber, 2001) 1224 252 51.7 �11.3

Mutant Candida boidinii FDH C262Vb (Slusarczyk et al., 2000; Felber, 2001) 3960 814 49.1 �13.9

Mutant Candida boidinii FDH C23S/C262Ab (Slusarczyk et al., 2000; Felber, 2001) 6430 1320 47.6 �15.4

Mutant Candida boidinii FDH C23S/C262A/E18N/K35R/E151D/R187S/F285Tb

(Slusarczyk et al., 2003)

131 27.9 58 �5

Mutant Candida boidinii FDH C23S/E151D/R178S/K306R/T315Nb

(Slusarczyk et al., 2003)

25.8 5.3 62 �1

wt-FDH Moraxella sp. (own data) 122 25 58 �5

wt-FDH M. vaccae N10 (Fedorchuk et al., 2002) 10.4 2.1 62 �1

Mutant M. vaccae N10 FDH E61K (Fedorchuk et al., 2002) 5.81 1.2 62.8 �0.2

wt-FDH Pseudomonas sp.101 (Fedorchuk et al., 2002) 4.86 1.0 63 0Mutant NAD+-specific FDH Pseudomonas sp.101 GAV (own data) 2.01 0.41 64.5 +1.5

Mutant NAD+-specific FDH Pseudomonas sp.101 T7 (own data) 0.097 0.020 68 +5

Mutant NADP+-specific FDH Pseudomonas sp.101 T5M9-10 (own data) 2.03 0.41 64.5 +1.5

Line for wild-type PseFDH is marked in bold because this enzyme used as a reference to show differences in stability.a Inactivation rate constants were calculated from data presented in this references supposing monomolecular mechanism of thermal denaturation.b kin for mutant CboFDHs were obtained by division of kin for wt-CboFDH at 55 8C by value of stabilization (destabilization) effect calculated from dependence in

Fig. 6 and Tm values from Felber (2001) (see text).

of first-order reactions. The dependence of the inactivation rate

constant kin on temperature T is described by the equation of the

transition state theory:

kin ¼kBT

he�

�DH 6¼

RT �DS 6¼R

�(1)

where T is the absolute temperature in K, kB and h the constants

of Boltzmann and Plank, respectively and R is the universal

thermodynamic constant. DH 6¼ and DS 6¼ are the activating

parameters of changes in enthalpy and entropy for the process

of enzyme thermal inactivation. This dependence can be

linearized using [ln(kin/T)] � 1/T plot. Values of DH 6¼ for

PseFDH and CboFDH determined from slope of correspond-

ing plots were 930 � 30 and 662 � 40 kJ/mol, respectively.

Higher DH 6¼ value for PseFDH compared to that for CboFDH

shows that the change in the rate constant upon temperature

rising for the bacterial enzyme is much bigger than that for

yeast FDH. At the same time upon temperature decrease the

inactivation rate constant for PseFDH will drop faster than one

for CboFDH, i.e. thermal stability of bacterial enzyme will also

grow faster than for CboFDH. MycFDH is a very good example

of the fact. DH 6¼ value 900 � 40 kJ/mol for this enzyme is

similar to one for PseFDH. At 62 8C MycFDH and the most

stable mutant CboFDH have the same values of Tm and kin

(Table 4), but at 55 8C MycFDH is 2.5-fold more stable as

mutant CboFDH due to higher DH 6¼ value (Table 4).

Table 4 presents the values for rate constants of thermal

inactivation of PseFDH and CboFDH at 55 8C. Based on the

data obtained by the other authors, we calculated the rate

constants for thermal inactivation of FDH from bacteria

Paracoccus sp.12-A (Shinoda et al., 2002), Thiobacillus

sp.KNK65MA (Nanba et al., 2003a) and A. aquaticus (Nanba

et al., 2003b). In addition, we give the values of Tm for these

enzymes. As seen from Table 4, FDH from Thiobacillus

sp.KNK65MA it the least stable enzyme. Taking into account

the results of mutagenesis of Cys residues in the other FDHs

(see above), one can suggest that the reason for such low

stability is the presence of Val and Ala residues in 255 and 288

positions in TbaFDH amino acid sequence instead of Cys

residues (Fig. 1).

Thermal denaturation studies of PseFDH, MorFDH,

CboFDH and SceFDH using DSC also prove PseFDH to be

the most thermostable enzyme among the known FDHs. The

details of these experiments will be published elsewhere. Fig. 4

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110102

Fig. 5. SDS-analytical electrophoresis of E. coli cell-free extract with mutant

PseFDH GAV before and after heat treatment at 60 8C (lines 1 and 2,

respectively).

shows melting curves for wt-CboFDH, wt-PseFDH and its

mutant with improved thermal stability (see below). In the case

of wt-PseFDH, heat of protein globule melting in the course of

transfer from native to denatured state is by 310 kJ/mol higher

that that for wt-CboFDH (2020 and 1710 kJ/mol for bacterial

and yeast enzymes, respectively).

5.2. Improvement of PseFDH thermal stability

To improve PseFDH thermal stability, the following appro-

aches were used: hydrophobization of a-helices (Rojkova et al.,

1999), increase in hydrophobicity of the protein globule,

optimization of electrostatic interactions (Fedorchuk et al.,

2002) and optimization of polypeptide chain conformation

(Serov and Tishkov, 2002; Serov et al., 2005). Selection of

mutation points was based on the X-ray analysis data and FDH

sequences alignment for the enzymes from different sources.

Note the stabilization effect for a single replacement was not

high, usually from 10 to 50%. However, in all cases, the

stabilization effect was additive (Rojkova et al., 1999; Serov

et al., 2005), i.e. the final value of the stabilization effect in the

multi-point mutant (nfin) was equal to the product of multi-

plication of individual stabilization effects nn for each single-

point mutation:

nfin ¼ n1 � n2 � n3 � � � � � nn

It was found that for many single and multi-point PseFDH

mutants (>90%) values DH6¼ are the same as for wild-type

enzyme, i.e. stabilizing effect of mutations was due to change of

DS 6¼ (Rojkova et al., 1999; Fedorchuk et al., 2002; Serov et al.,

2005). In some cases, the introduced replacements (such as

Lys61Pro) had no effect on the thermal stability, but improved

the stability in high ionic strength solutions (Fedorchuk et al.,

2002).

Combination of seven best mutations resulted in production

of PseFDH T7 mutant with a 50-fold lower thermal inactivation

rate constant compared to wt-PseFDH (Table 4), and the

melting temperature in DCS experiments increased by 6.68(Fig. 4). The mutations improving operational and thermal

stability were combined in PseFDH GAV mutant. The

combination compensated the decrease in thermal stability

resulting from Cys replacement, and in addition, improved the

overall thermal stability 2.5-fold compared to wt-PseFDH

(Fig. 4). Moreover, the mutant showed two-fold increase in

affinity for NAD+ (InnoTech MSU, 2006).

The construction of mutant PseFDHs with increased

thermal stability allowed the step of heat treatment of cell-

free extract to be introduced into the purification protocol for

the recombinant enzyme. Incubation of cell-free extract at

60 8C for 20–30 min increases the purity of the PseFDH GAV

preparation from 50 to 80–85% without any loss of enzyme

activity (Fig. 5).

5.3. Improvement of CboFDH thermal stability

An improvement of thermal stability of CboFDH was obv-

iously a more complicated task because the simultaneous

replacement of two Cys residues in each subunit resulted in a

significant decrease in the enzyme thermal stability (35–94-

fold) (Slusarczyk et al., 2003) compared to that for PseFDH

(10-fold). To increase the thermal stability of Cys23Ser and

Cys23Ser/Cys262Ala CboFDH mutants, the method of

‘‘directed evolution’’ has been applied (Slusarczyk et al.,

2003). The screening of two libraries, 200,000 clones each,

yielded three clones derived from Cys23Ser/Cys262Ala double

mutant and seven clones derived from Cys23Ser CboFDH

single mutant. The stabilizing replacements increased Tm

values by 10–118 compared to the original mutants (from 52 to

62, and from 47 to 58 8C for Cys23Ser and Cys23Ser/

Cys262Ala CboFDH, respectively) (Slusarczyk et al., 2003).

As it was mentioned above, Tm values do not give

quantitative assessment of the stabilization effects. To

determine the quantitative parameters for the stabilization

effects we employed the transition state theory (see above). By

analogy with PseFDH, we assumed that the introduction of

mutations into CboFDH did not change DH 6¼ value. If this is the

case, the dependence of the inactivation rate constant kin,

determined for the set of mutants at the same temperature, will

be linear in a [ln(kin/Tm)] � 1/Tm plot, where Tm is expressed in

Kelvin. The work (Felber, 2001) presents the values for half-life

periods t1/2 for wt-CboFDH and its Cys mutants at 50 8C. The

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 103

Fig. 6. Dependence of half-life periods t1/2 for wt-CboFDH and its Cys mutants

at 50 8C on Tm in coordinates [ln(1/(t1/2 � Tm)] � 1/Tm. Values of t1/2 and Tm

were taken from Felber (2001).

Fig. 7. pH-profiles for Km for NADP+ for the first generation of mutant NADP+-

specific PseFDH M9 and the second generation mutants PseFDH M9-10 and

PseFDH M9-11. Reproduced with permission from InnoTech MSU (2005).

t1/2 value is reciprocally proportional to kin, therefore, a [ln(1/

(t1/2 � Tm)] � 1/Tm plot should be linear as well (Tm has to be

expressed in grad K). Fig. 6 demonstrates that a true linear

dependence (error < 3%, correlation coefficient R > 0.999)

between half-life times of inactivation at 50 8C and Tm is

observed. It was the linear dependence that allowed us to get

correlation between kin and Tm and quantitatively evaluate the

role of individual mutations in enzyme stabilization (Table 1).

Among all mutations introduced, the biggest effects were

observed for replacements Glu151Asp, Arg178Ser, Arg178Gly

4 Asp149Glu (the stabilization effects were 9.7, 3.4, 2.3 and

2.15, respectively). We want to highlight the Glu151Asp

mutation, which contributed most to the stabilization. This

replacement can be predicted from alignment of FDHs amino

acid sequences. Among 20 FDHs from yeast and fungi only

CboFDH has Glu residue in position 151. In other cases, there

are 15 Asp and 4 Asn residues in this position. In equivalent

position, bacterial FDHs have only Asp residue (Fig. 1). At the

same time Arg in position 178 (numeration according CboFDH

aa sequence) is absolutely conservative residue for FDHs from

yeast and fungi (Fig. 1). The analysis of Asp149, Glu151 and

Arg178 positions in the model structure of CboFDH shows their

location in region of intersubunit contacts. The stabilization

effect of the other replacements, e.g. Glu18Asp, Lys35Arg,

Phe285Tyr, Lys306Arg, Thr315Asn and Lys356Glu, did not

increase more than 1.5-fold (Table 1).

Thus, the problem of CboFDH stabilization has been

successfully solved. The value of thermal stability of Cys23Ser

and Cys23Ser/Cys262Ala CboFDH mutants was increased 48-

and 56-fold, respectively, and if compared to the wild-type

enzyme, 7.1- and 1.6-fold, respectively.

The data on the role of the other amino acid residues in

FDHs from C. boidinii and C. methylica are illustrated in

Table 1.

6. Change of coenzyme specificity

Formate dehydrogenase is a highly specific enzyme with

respect to NAD+ (Tishkov and Popov, 2004). The data on

coenzyme preference ðkcat=KmÞNADþ=ðkcat=KmÞNADPþfor

FDHs from Pseudomonas sp.101, C. methylica and S.

cerevisiae were presented in Gul-Karaguler et al. (2001) and

Serov et al. (2002). The analysis of kinetic properties of plant

FDHs from A. thaliana and soya G. max expressed in E. coli

cells in our laboratory shows the similarity in their coenzyme

preference with that of PseFDH.

Mutant PseFDH with coenzyme specificity changed from

NAD+ to NADP+ was prepared in 1993. The enzyme was

successfully used in synthesis of chiral alcohols and e-lactones

using alcohol dehydrogenases and cyclohexanone monooxy-

genases (Seelbach et al., 1996; Rissom et al., 1997; Schwarz-

Linek et al., 2001). Unfortunately, contrary to NAD+-specific

PseFDH, which has Km for NAD+ unchanged in pH range

6.0–9.0 (Mesentsev et al., 1997), the first generation of NADP+-

dependent mutant enzymes (version PseFDH T5M8) demon-

strated the constant value of Km for NADP+ only in the pH range

of 6.0–7.4 (InnoTech MSU, 2005). At pH � 8.0, the KNADPþm

value increases 10-fold and higher. Recently, new NADP+-

specific formate dehydrogenase PseFDH T5M9-10 have been

prepared and this mutant enzyme has extended pH optimum for

KNADPþm (pH range 6.0–9.0) (Fig. 7) (InnoTech MSU, 2005).

The analysis of experiments resulting in the change of

coenzyme specificity of FDHs from Pseudomonas sp.101, C.

methylica and S. cerevisiae was reviewed earlier (Serov et al.,

2002; Tishkov and Popov, 2004). Additional information about

new NADP+-specific PseFDHs can be found in InnoTech MSU

(2005). Herein, we will discuss the recently published results on

the change in coenzyme specificity of CboFDH (Rozzell et al.,

2004). CboFDH mutant active with NADP+ was prepared by

directed evolution based on the Asp195Ser mutants described

in Gul-Karaguler et al. (2001). The resultant forms were

CboFDH double D195S/Y196H and triple D195S/Y196H/

K356T mutants. The activity of single, double and triple

CboFDH mutants with NAD+ decreased (1.5, 1.3 and 1.3 U/mg,

respectively) as compared to the activity wt-CboFDH (2.2 U/

mg). Introduction of replacements resulted in the increase of

enzyme activity with NADP+ from 0.0013 for wild-type

enzyme to 0.083, 0.19 and 0.36 U/mg for single, double and

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110104

Fig. 8. Orientation of Asp195, Tyr196 and Lys356 towards NAD+ in model

structure of binary complex (CboFDH-NAD+). Picture was created using

WebLab ViewerPro 3.7 software (Molecular Simulations Inc.).

triple mutants, respectively. Unfortunately, there is no data

about the values of Km for NADP+ and formate. The activity of

enzymes was measured at room temperature. If recalculated for

30 8C, the activity of best mutant CboFDH with NADP+ should

be ca. 1.0 U/mg. This value is 2.5-fold lower if compared to the

activity of NADP+-specific PseFDH (Serov et al., 2002;

InnoTech MSU, 2005).

The analysis of holo-CboFDH model structure shows that

Asp195 and Lys356 form hydrogen bonds with 20- and 30-OH

groups of adenosine ribose (Fig. 8). The Asp195Ser replace-

ment results in the removal of the negative charge, and the

Lys356Thr replacement, probably, provides additional room for

the phosphate group. Note, PseFDH contains His379 in the

position equivalent to Lys356 in CboFDH (Fig. 1). One may

suggest that the positively charged His residue with lesser

volume of the side chain compared to that of Lys, could

participate in NADP+ binding.

Table 5

Expression of formate dehydrogenases in E. coli cells

Source of gene Level o

(% of so

Pseudomonas sp.101 NAD+-specifica 50–55

Pseudomonas sp.101 NADP+-specific (Tishkov et al., 1999) 50–55

M. vaccae N10 (Yamamoto et al., 2005) 30–35

M. vaccae N10a 50–55

Moraxella sp.a 50–55

Paracoccus sp.12-A (Shinoda et al., 2002) 12

Ancylobacter aquaticus (Nanba et al., 2003b) 44

Thiobacillus sp.KNK65MA (Nanba et al., 2003a) n.d.

Candida methylica (Allen and Holbrook, 1995) 15

Candida boidinii (Slusarczyk et al., 2000; Labrou et al., 2000) 18

Candida boidinii (Felber, 2001) 15

Candida boidinii (Rozzell et al., 2004) 20–40

Candida boidiniia 35–40

Saccharomyces cerevisiae (Serov, 2002) 30–35

Soya G. maxa 25–30

Arabidopsis thalianaa 30–35

a Own data.b n.d., no data.c Personal communication of Dr. T. Daussmann.

In accordance with our model of holo-CboFDH structure,

Tyr196 residue is not oriented towards adenosine ribose

(Fig. 8), while in the model structure for SceFDH, this residue

forms a hydrogen bond with 30-OH group of adenosine ribose

(Serov et al., 2002). Most likely, Tyr196His replacement

provides an additional positive charge in the coenzyme-binding

domain, necessary to compensate the negative charge of 30-phosphate group of NADP+.

Thus, in the result of three replacements only, the authors

were able to get CboFDH mutant with sufficiently high activity

towards NADP+. This enzyme was used for NADPH regene-

ration in (S)-ethyl 4-chloro-3-hydroxybutanoate production

with NADP+-specific ketoreductase (Rozzell et al., 2004).

7. Expression of FDH genes in E. coli cells

Production of individual enzymes even partially purified is a

costly process. Therefore, to lower the production cost, one

constructs recombinant strains superproducing the target

enzyme. Currently, FDH from bacteria, Pseudomonas sp.101

(Tishkov et al., 1991, 1999), M. vaccae N10 (Fedorchuk et al.,

2002; Yamamoto et al., 2005), Moraxella sp., Hyphomicrobium

strain JT-17 (FERM P-16973) (Mitsunaga et al., 2000),

Paracoccus sp.12-A (Shinoda et al., 2002), Thiobacillus

sp.KNK65MA (Nanba et al., 2003a), A. aquaticus (Nanba

et al., 2003b), yeast C. methylica (Allen and Holbrook, 1995),

C. boidinii (Sakai et al., 1997; Slusarczyk et al., 2000; Labrou

et al., 2000; Felber, 2001) and baker’s yeast S. cerevisiae (Serov

et al., 2002; Serov, 2002) are successfully cloned and expressed

in E. coli. In this laboratory, plant FDHs, from soybean and A.

thaliana (the genes were kindly provided by Profs. N. Labrou

and J. Markwell, respectively) have been expressed in E. coli as

active enzymes. Noteworthy, the first plant FDH genes were

cloned 8–12 years ago, but there were no data reported on their

expression in E. coli in active and soluble form. Some details

f expression

luble E. coli proteins)

Inducer Production scale per run

Lactose Megaunits

Lactose Hundred kilounits

IPTG n.d.b

Lactose Dozen kilounits

Lactose Hundred kilounits

IPTG n.d.

IPTG n.d.

IPTG n.d.

IPTG n.d.

IPTG n.d.

IPTG Megaunitsc

n.d n.d.

Lactose Dozen kilounits

Lactose Dozen kilounits

Lactose Dozen kilounits

Lactose Dozen kilounits

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 105

about expression of FDHs in E. coli cells are presented in

Table 5.

In all above experiments, FDH was synthesized in E. coli as

active enzyme. The level of expression varied from 12–15 to

50–55% of the total soluble E. coli protein. The lower

expression level for plant and yeast FDHs is likely to be

caused by the presence of Arg codons rare for E. coli, i.e.

AGA and AGG. To improve yield of recombinant CboFDH,

Rozzell et al. (2004) optimized CboFDH gene sequence for E.

coli codon usage and synthesized the modified gene. In

addition, a Gly residue has been added just after the N-

terminal Met. As for PseFDH, the gene sequence had no effect

on the expression level, nevertheless, the optimization of a

number of codons resulted in a two-fold increase in the

enzyme biosynthesis rate.

Commercial production of recombinant FDH was developed

for the enzymes from C. boidinii and Pseudomonas sp.101. For

PseFDH, the maximum yield of the enzyme was ca. 35 kU l�1

of cultural medium. Fermentation was performed in a fed-batch

mode during 19 h at 25 8C in the absence of antibiotics. Lactose

served as an inducer. Time/space yield was ca. 1850 U l�1 h�1,

and the enzyme specific activity in the cell-free extract was

equal to 4.5–5.5 U/mg of protein. Introduction of mutations

improving thermal and chemical stability of PseFDH, had no

effect on cultivation results.

Optimization of large scale preparation of recombinant

CboFDH is described in Felber (2001). Fermentation was

performed for 36–41 h at 30 8C with subsequent IPTG

induction. The maximum yield was ca. 60 kU l�1, time/space

yield 1600 U l�1 h�1 and the specific activity 0.8–1.0 U/mg.

These results were the same for the wild-type and C23S,

C262V, C23S/C262V and C23S/C262A mutant enzyme forms.

Accounting for the improved catalytic activity of CboFDH,

from 6.0 to 9.1 U/mg (Felber, 2001), the enzyme yield can be

expected up to 90 kU l�1.

As could be seen from the data provided, process of

recombinant mutant CboFDH production gives higher time/

space yield of active enzyme than PseFDH. However, the

prolonged duration and lower expression level of CboFDH

compared to those for PseFDH results in higher production costs:

preparation of one activity unit of CboFDH requires 8–10-fold

higher glucose expense than preparation of one activity unit of

PseFDH; the lower temperature of PseFDH cultivation and use of

a cheaper inducer, lactose instead of IPTG, also helps to reduce

the production costs. In addition, the higher content of PseFDH in

the biomass (50–55% of the total soluble protein compared to

15% for CboFDH) significantly simplifies, and therefore, lowers

the reagent consumption and the cost of purification.

8. Alternative enzymes for NAD(P)H regeneration

Many enzymes were tested and used for NAD(P)H

regeneration in processes of enzyme chiral synthesis and

formate dehydrogenase is still the gold standard in this area

(van der Donk and Zhao, 2003). Detailed information about

most successful examples can be found in review (Wichmann

and Vasic-Racki, 2005). Here, we will shortly describe two

alternatives to FDH enzymes, glucose dehydrogenase and

phosphite dehydrogenase. The first one is already widely used

in practice. Phosphite dehydrogenase is a newcomer in this area

and looks a promising candidate for coenzyme regeneration.

8.1. Glucose dehydrogenase

Gluconolactone, product of reaction catalyzed by glucose

dehydrogenase, GDH, is spontaneously hydrolysed to gluconic

acid. This makes the overall reaction irreversible and enables to

use GDH for cofactor regeneration. Majority of GDHs show

dual coenzyme specificity, however with a preference towards

one of the coenzyme forms, either NAD+ or NADP+. Several

isoenzymes of GDH can be found in one strain. For example,

strain Bacillus megaterium IAM1030 harbours four GDH

isoenzymes. Two isoenzymes prefer NAD+ as a coenzyme,

while the other two as NADP+ (Nagao et al., 1992). Wide

abundance of GDH in nature enables to search for the enzymes

showing maximal activity at extreme conditions, e.g. under high

temperatures (Bright et al., 1993), acidic pH (Angelov et al.,

2005) or very high salt concentration (Bonete et al., 1996). A

wide number of commercial GDH preparations are available

from various sources (for example, see product catalogs of

Biocatalytics Inc. and Julich Chiral Solutions). Higher specific

activity of GDH (20–100 U/mg) compared to FDH (2.5–10 U/

mg) is the clear advantage of GDH-based NAD(P)H regeneration

system over FDH-based. Glucose and ammonium formate have

similar prices, but reducing equivalent capacity (REC) of glucose

is about four-fold less. Reduction of one mole of NAD(P)H

requires 172 g of glucose and only 45 g of formate (in calculation

of REC we took into account only molecular mass of formate ion

and did not consider molecular mass of ammonium ion because

in the reaction it acts only as a buffer component). The other

disadvantage of GDH-based regeneration systems results from

the necessity to purify the end-product from gluconic acid.

All microorganisms have a system of active transport of

glucose (as well as formate) inside the cell. Therefore, during the

last years GDH is actively used for coenzyme regeneration in the

processes where whole cells are utilized as biocatalysts (Endo

and Koizumi, 2001; Kataoka et al., 2003, 2004). To produce such

a biocatalyst the main enzyme and GDH can be expressed in two

separate strains (Liu et al., 2005; Xu et al., 2005), as well as co-

expressed in one strain (Kataoka et al., 1997; Wada et al., 2003;

Yun et al., 2005). In this whole-cell approach even intracellular

pool of NAD(P)+ is enough to achieve the necessary level of

cofactor regeneration (Ishige et al., 2005) and gluconic acid,

product of glucose oxidation, can be further utilized by the cell as

a carbon source. Similar recombinant E. coli strains, co-

expressing formate dehydrogenase and two or more enzymes,

were also constructed (Galkin et al., 1997a,b; Ernst et al., 2005).

8.2. Phosphite dehydrogenase

Phosphite dehydrogenase from Pseudomonas stutzeri

WM88 (PTDH) has been recently proposed as an alternative

enzyme for NAD(P)H regeneration (Vrtis et al., 2005; Relyea

and van der Donk, 2005). The enzyme catalyzes reaction of

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110106

phosphite oxidation to phosphate with corresponding reduction

of NAD+ to NADH. The reaction is irreversible and can be used

for coenzyme regeneration. Wild-type PTDH has the same kcat

value as PseFDH, 7.3 s�1 (Costas et al., 2001). Km value for

NAD+ does not depend on pH, while kcat=Kphosphitem value shows

a maximum in the pH range of 7.0–7.6. Above and below this

range, dehydrogenase activity drops off steeply (Relyea et al.,

2005). Primordial PTDH exhibits poor thermal stability.

Temperature optimum of activity for the wild-type enzyme

is as low as 35 8C (Costas et al., 2001). The random

mutagenesis generated PTDH preparations showing an

improved thermal stability, approximately 2.5-fold better than

that of wild-type CboFDH (Johannes et al., 2005). Due to this

fact the mutant PTDH was more effective than wt-CboFDH in

the synthesis of tert-L-leucine (Johannes et al., 2005). In future

it would be also interesting to compare mutant PTDH with

PseFDH GAV, which is commercially available since 2001. At

50 8C PseFDH GAV has 100-fold higher thermal stability

compared to wt-CboFDH and consequently 40-fold compared

to current version of mutant PTDH.

Wild-type PTDH is NAD+-specific, but exhibits rather high

activity with NADP+. Value of kcat with NADP+ is only 50% of

kcat value with NAD+ (Woodyer et al., 2003). Site-directed

mutagenesis resulted in production of NADP+-specific PTDH

(Woodyer et al., 2005). Its application to NADPH regeneration

in the reaction of xylitol production from xylose catalyzed by

xylose reductase showed a four-fold increase in the rate of

synthesis of the final product as compared to the NADP+-

dependent PseFDH (version T5M8) and wt-PTDH. One has to

admit, that the use of reaction of xylitol synthesis as a

reference to compare the efficiency of NADPH regeneration is

not the best choice, as the main enzyme, xylose reductase, is

apparently the only one that is reported to be inhibited by

formate (Neuhauser et al., 1998). Therefore, the lower

reaction rate of xylitol production in the system using

NADP+-specific PseFDH for coenzyme regeneration, in this

particular case, can be attributed to the xylose reductase

inhibition itself.

Phosphite-ion has about two-fold better reducing equivalent

capacity in comparison with glucose and is two-fold inferior

compared to formate-ion. An advantage of PTDH over other

dehydrogenases is the ease with which it can be used for the

preparation of deuterated compounds. Preparation of deuter-

Table 6

Costs of formate dehydrogenases from different companiesa

Enzyme Biocatalytics Inc.

US$ per

1000 U

US$ per

10,000 U

NAD+-specific from C. boidiniib 390 1950

NAD+-specific from Pseudomonas sp.101c – –

NADP+-specific from Pseudomonas sp.101d – –

a Prices for December 2005.b Recombinant wild-type enzyme.c Mutant PseFDH GAV with increased chemical and thermal stability and improd Mutant enzyme with extended pH-optimum for NADP+ and increased chemica

ated phosphite from normal substrate can be performed by

incubation in D2O at pH 2.0 followed by liophylization (Vrtis

et al., 2005). Therefore, the price of deuterated phosphite is

lower compared to prices for deuterated formate, alcohols and

particularly glucose.

9. Conclusion

Chiral compounds synthesis is a rapidly growing area in

biotechnology. NAD(P)+-dependent dehydrogenases and reduc-

tases are the most effective biocatalysts for such type of

processes. First, the use of these enzymes allows optically active

molecules to be produced from non-chiral substrates. Second, the

reactions catalyzed by dehydrogenases are extremely stereo-

specific (LaReau and Anderson, 1989; Weinhold et al., 1991),

and the yield of the final product may reach 100%, while the

processes based on kinetic resolution of racemic mixtures can

provide the theoretic yield of 50%. The main disadvantage of the

dehydrogenase-catalyzed process is the high cost of NADH and

especially NADPH. The development of regeneration systems

for reduced coenzymes and methods for their retention in

bioreactors made great impact to use dehydrogenases in

synthesis of chiral alcohols from ketones with alcohol

dehydrogenases and natural and artificial amino acids with

keto-acid dehydrogenases as well as to use monooxygenases for

hydroxylation and epoxidation. The processes were reviewed in

detail in Liese and Villela (1999), Liese (2005) and Wichmann

and Vasic-Racki (2005). In the last decade, the improvement of

formate dehydrogenase properties and development of large

scale production with recombinant E. coli strains significantly

reduced the cost of FDH in the overall production cost of target

compounds. Production of NADP+-specific FDH opened the

possibility for its application for the purposes of NADPH

regeneration (Seelbach et al., 1996; Rissom et al., 1997;

Schwarz-Linek et al., 2001; Maurer et al., 2003). Recombinant

CboFDH is available in large volumes from Julich Chiral

Solutions (Julich Fine Chemicals before January 2006) in Europe

and from Biocatalytics Inc. in the USA (Table 6). Unfortunately,

all mutations providing improvement of CboFDH properties are

covered by patents and at the moment only wild-type enzyme

is commercially available. Recombinant PseFDH GAV with

improved chemical and thermal stability is available from Julich

Chiral Solutions and Innovations and High Technologies MSU

Julich Chiral Solutions

(Julich Fine Chemicals)

Innovations and High

Technologies MSU

s per

1000 U

s per

10,000 U

s per

1000 U

s per

10,000 U

290 750 – –

– 950 150 570

680 3400 420 2000

ved affinity for NAD+.

l and thermal stability.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 107

(InnoTech MSU). Mutant NADP+-specific PseFDH is also

offered by these companies (Table 6).

In our opinion, if trying to predict the further development in

this research area, the NADH regeneration with high purity

enzymes will get broader commercial impact than that of

NADPH regeneration. This prediction is based on high cost and

low stability of NADP+ compared to NAD+. The NAD+-

dependent enzyme can be derived from the corresponding

NADP+-specific analog. As an example of the concept, we cite

here the successful change of coenzyme specificity of reductase

in cytochrome P-450 monooxygenase from B. megaterium

(P450 BM-3) (Urlacher and Schmid, 2004). Wild-type and

mutant monooxygenase were successfully used for hydroxyla-

tion of poorly soluble compounds in combination with NADP+-

and NAD+-specific PseFDHs in two-phase water–cyclohexane

system (Hofstetter et al., 2004; Maurer et al., 2003, 2005;

Urlacher et al., 2005).

Another prospective trend for FDH application research is

metabolic engineering of recombinant strains. Expression of

FDH gene in a recombinant strain gives an additional supply of

intracellular NADH and NADPH when growing in the

presence of formate. This helps to release other metabolic

pathways producing NADH or NADPH and redirect them to

the synthesis of the target product (Berrios-Rivera et al.,

2002a,b, 2004; San et al., 2002; Kaup et al., 2004; Sanchez

et al., 2005).

In conclusion, we think that the existence of numerous FDH

genes from various sources opens new horizons in improve-

ment of enzyme properties with gene shuffling. In the first turn,

the attention will be paid to further increase in catalytic activity

because FDH is still a ‘‘slow’’ enzyme compared to other

dehydrogenases.

Acknowledgments

Authors thanks Dr. I. Gazaryan for help in manuscript

preparation, Dr. S. Felber for presentation of his Ph.D.

dissertation. This work was supported by grants from Russian

Foundation for Basic Research (project a05-04-49073), NATO

(grant no. LST.CLG 977839) and Russian Federal Agency for

Science and Innovations (FASI contract 02.435.11.3005).

References

Allen, S.J., Holbrook, J.J., 1995. Isolation, sequence and overexpression of the

gene encoding NAD-dependent formate dehydrogenase from the methylo-

trophic yeast Candida methylica. Gene 162, 99–104.

Angelov, A., Futterer, O., Valerius, O., Braus, G.H., Liebl, W., 2005. Properties

of the recombinant glucose/galactose dehydrogenase from the extreme

thermoacidophile, Picrophilus torridus. FEBS J. 272, 1054–1062.

Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K.-I., Oguchi, A., Nagai,

Y., Iwama, N., Asano, K., Naimi, T., 2002. Genome and virulence deter-

minants of high virulence community-acquired MRSA. Lancet 359, 1819–

1827.

Barnett, M.J., Fisher, R.F., Jones, T., Komp, C., Abola, A.P., Barloy-Hubler, F.,

Bowser, L., Capela, D., Galibert, F., Gouzy, J., Gurjal, M., Hong, A., Huizar,

L., Hyman, R.W., Kahn, D., Kahn, M.L., Kalman, S., Keating, D.H., Palm,

C., Peck, M.C., Surzycki, R., Wells, D.H., Yeh, K.C., Davis, R.W.,

Federspiel, N.A., Long, S.R., 2001. Nucleotide sequence and predicted

functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc.

Natl. Acad. Sci. U.S.A. 98, 9883–9888.

Beja, O., Aravind, L., Koonin, E.V., Suzuki, M.T., Hadd, A., Nguyen, L.P.,

Jovanovich, S.B., Gates, C.M., Feldman, R.A., Spudich, J.L., Spudich, E.N.,

DeLong, E.F., 2000. Bacterial rhodopsin: evidence for a new type of

phototrophy in the sea. Science 289, 1902–1906.

Berrios-Rivera, S.J., Bennett, G.N., San, K.Y., 2002a. The effect of increasing

NADH availability on the redistribution of metabolic fluxes in Escherichia

coli chemostat cultures. Metab. Eng. 4, 230–237.

Berrios-Rivera, S.J., Bennett, G.N., San, K.Y., 2002b. Metabolic engineering of

Escherichia coli: increase of NADH availability by overexpressing an

NAD+-dependent formate dehydrogenase. Metab. Eng. 4, 217–229.

Berrios-Rivera, S.J., Sanchez, A.M., Bennett, G.N., San, K.Y., 2004. Effect of

different levels of NADH availability on metabolite distribution in Escher-

ichia coli fermentation in minimal and complex media. Appl. Microbiol.

Biotechnol. 65, 426–432.

Bommarius, A.S., Schwarm, M., Stingl, K., Kottenhahn, M., Huthmacher, K.,

Drauz, K., 1995. Synthesis and use of enantiometrically pure tert-leucine.

Tetrahedron-Asymmetry 6, 2851–2888.

Bonete, M.J., Pire, C., Lorca, F.I., Camacho, M.L., 1996. Glucose dehydro-

genase from the halophilic Archaeon Haloferax mediterranei: enzyme

purification, characterisation and N-terminal sequence. FEBS Lett. 383,

227–229.

Bright, J.R., Byrom, D., Danson, M.J., Hough, D.W., Towner, P., 1993. Cloning,

sequencing and expression of the gene encoding glucose dehydrogenase

from the thermophilic archaeon Thermoplasma acidophilum. Eur. J. Bio-

chem. 211, 549–554.

Burton, S.G., 2003. Oxidizing enzymes as biocatalysts. Trends Biotechnol. 21,

543–549.

Cazalet, C., Rusniok, C., Bruggemann, H., Zidane, N., Magnier, A., Ma, L.,

Tichit, M., Jarraud, S., Bouchier, C., Vandenesch, F., Kunst, F., Etienne, J.,

Glaser, P., Buchrieser, C., 2004. Evidence in the Legionella pneumophila

genome for exploitation of host cell functions and high genome plasticity.

Nat. Genet. 36, 1165–1173.

Chien, M., Morozova, I., Shi, S., Sheng, H., Chen, J., Gomez, S.M., Asamani,

G., Hill, K., Nuara, J., Feder, M., Rineer, J., Greenberg, J.J., Steshenko, V.,

Park, S.H., Zhao, B., Teplitskaya, E., Edwards, J.R., Pampou, S., Georghiou,

A., Chou, I.-C., Iannuccilli, W., Ulz, M.E., Kim, D.H., Geringer-Sameth, A.,

Goldsberry, C., Morozov, P., Fischer, S.G., Segal, G., Qu, X., Rzhetsky, A.,

Zhang, P., Cayanis, E., De Jong, P.J., Ju, J., Kalachikov, S., Shuman, H.A.,

Russo, J.J., 2004. The genomic sequence of the accidental pathogen

Legionella pneumophila. Science 305, 1966–1968.

Colas des Francs-Small, C., Ambard-Bretteville, F., Small, I.D., Remy, R.,

1993. Identification of a major soluble protein in mitochondria from

nonphotosynthetic tissues as NAD-dependent formate dehydrogenase. Plant

Physiol. 102, 1171–1177.

Costas, A.M., White, A.K., Metcalf, W.W., 2001. Purification and character-

ization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri

WM88. J. Biol. Chem. 276, 17429–17436.

Davidson, D.C., 1951. Studies on plant formic dehydrogenase. Biochem. J. 49,

520–526.

Demchenko, A.P., Rusyn, O.I., Egorov, A.M., Tishkov, V.I., 1990. The solvent

effects on the kinetics of bacterial formate dehydrogenase. Biochim.

Biophys. Acta 1039, 290–296.

Dujon, B., Sherman, D., Fischer, G., Durrens, P., Casaregola, S., Lafontaine, I.,

De Montigny, J., Marck, C., Neuveglise, C., Talla, E., Goffard, N., Frangeul,

L., Aigle, M., Anthouard, V., Babour, A., Barbe, V., Barnay, S., Blanchin, S.,

Beckerich, J.M., Beyne, E., Bleykasten, C., Boisrame, A., Boyer, J.,

Cattolico, L., Confanioleri, F., De Daruvar, A., Despons, L., Fabre, E.,

Fairhead, C., Ferry-Dumazet, H., Groppi, A., Hantraye, F., Hennequin, C.,

Jauniaux, N., Joyet, P., Kachouri, R., Kerrest, A., Koszul, R., Lemaire, M.,

Lesur, I., Ma, L., Muller, H., Nicaud, J.M., Nikolski, M., Oztas, S., Ozier-

Kalogeropoulos, O., Pellenz, S., Potier, S., Richard, G.F., Straub, M.L.,

Suleau, A., Swennen, D., Tekaia, F., Wesolowski-Louvel, M., Westhof, E.,

Wirth, B., Zeniou-Meyer, M., Zivanovic, I., Bolotin-Fukuhara, M., Thierry,

A., Bouchier, C., Caudron, B., Scarpelli, C., Gaillardin, C., Weissenbach, J.,

Wincker, P., Souciet, J.L., 2004. Genome evolution in yeasts. Nature 430,

35–44.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110108

Endo, T., Koizumi, S., 2001. Microbial conversion with cofactor regeneration

using genetically engineered bacteria. Adv. Synth. Catal. 343, 521–526.

Ernst, M., Kaup, B., Muller, M., Bringer-Meyer, S., Sahm, H., 2005. Enantio-

selective reduction of carbonyl compounds by whole-cell biotransforma-

tion, combining a formate dehydrogenase and a (R)-specific alcohol

dehydrogenase. Appl. Microbiol. Biotechnol. 66, 629–634.

Fedorchuk, V.V., Galkin, A.G., Yasny, I.E., Kulakova, L.B., Rojkova, A.M.,

Filippova, A.A., Tishkov, V.I., 2002. Influence of interactions between

amino acid residues 43 and 61 on thermal stability of bacterial formate

dehydrogenases. Biochemistry (Mosc.) 67, 1145–1151.

Felber, S., 2001. Optimierung der NAD-abhangigen Formiatdehydrogenase aus

Candida boidinii fur den Einsatz in der Biokatalyse. Ph.D. Thesis. Heinrich-

Heine University of Duesseldorf. URL: http://diss.ub.uni-duesseldorf.de/

ebib/diss/file?dissid=78.

Filippova, E.V., Polyakov, K.M., Tikhonova, T.V., Stekhanova, T.N., Boiko,

K.M., Popov, V.O., 2005. Structure of a new crystal modification of the

bacterial NAD+-dependent formate dehydrogenase with a resolution of

2.1 A Crystallogr. Rep. 50, 796–800.

Filippova, E.V., Polyakov, K.M., Tikhonova, T.V., Stekhanova, T.N., Boiko,

K.M., Sadykhov, E.G., Tishkov, V.I., Popov, V.O., 2006. Crystal structure of

NAD+-dependent formate dehydrogenase from Pseudomonas sp.101 in

complex with formate. Crystallogr. Rep. 51, N2.

Galkin, A., Kulakova, L., Tishkov, V., Esaki, N., Soda, K., 1995. Cloning of

formate dehydrogenase gene from a methanol-utilizing bacterium Myco-

bacterium vaccae N10. Appl. Microbiol. Biotechnol. 44, 479–483.

Galkin, A., Kulakova, L., Yoshimura, T., Soda, K., Esaki, N., 1997a. Synthesis

of optically active amino acids from alpha-keto acids with Escherichia coli

cells expressing heterologous genes. Appl. Environ. Microbiol. 63, 4651–

4656.

Galkin, A., Kulakova, L., Yamamoto, H., Tanizawa, K., Tanaka, H., Esaki, N.,

Soda, K., 1997b. Conversion of alpha-keto acids to D-amino acids by

coupling of four enzyme reactions. J. Ferment. Bioeng. 83, 299–300.

Gul-Karaguler, N., Sessions, R.B., Clarke, A.R., Holbrook, J., 2001. A single

mutation in the NAD-specific formate dehydrogenase from Candida methy-

lica allows the enzyme to use NADP. Biotechnol. Lett. 23, 283–287.

Hofstetter, K., Lutz, J., Lang, I., Witholt, B., Schmid, A., 2004. Coupling of

biocatalytic asymmetric epoxidation with NADH regeneration in organic–

aqueous emulsions. Angew. Chem. Int. Ed. 43, 2163–2166.

Hummel, W., Kula, M.R., 1989. Dehydrogenases for the synthesis of chiral

compounds. Eur. J. Biochem. 184, 1–13.

Hwang, L., Hocking-Murray, D., Bahrami, A.K., Andersson, M., Rine, J., Sil,

A., 2003. Identifying phase-specific genes in the fungal pathogen Histo-

plasma capsulatum using a genomic shotgun microarray. Mol. Biol. Cell 14,

2314–2326.

Innovations and High Technologies MSU (InnoTech MSU Ltd.), 2005. NADP+-

specific formate dehydrogenase from Pseudomonas sp.101. Product

description. URL: http://www.innotech-msu.com/products/NADPFDH.pdf.

Innovations and High Technologies MSU (InnoTech MSU Ltd.), 2006. Mutant

formate dehydrogenase from Pseudomonas sp.101 with improved affinity

for NAD+ and increased chemical and thermal stability (version PseFDH

GAV) overexpressed in E. coli. Product description. URL: http://www.in-

notech-msu.com/products/NADFDHGAV.pdf.

Ishige, T., Honda, K., Shimizu, S., 2005. Whole organism biocatalysis. Curr.

Opin. Chem. Biol. 9, 174–180.

Johannes, T.W., Woodyer, R.D., Zhao, H., 2005. Directed evolution of a

thermostable phosphite dehydrogenase for NAD(P)H regeneration. Appl.

Environ. Microbiol. 71, 5728–5734.

Jones, T., Federspiel, N.A., Chibana, H., Dungan, J., Kalman, S., Magee, B.B.,

Newport, G., Thorstenson, Y.R., Agabian, N., Magee, P.T., Davis, R.W.,

Scherer, S., 2004. The diploid genome sequence of Candida albicans. Proc.

Natl. Acad. Sci. U.S.A. 101, 7329–7334.

Julich Fine Chemicals, 2005. Product Catalog.

Karaguler, N.G., Sessions, R.B., Moreton, K.M., Clarke, A.R., Holbrook, J.J.,

2004. Estimating the energetic contribution of hydrogen bonding to the

stability of Candida methylica formate dehydrogenase by using double

mutant cycle. Biotechnol. Lett. 26, 1137–1140.

Kataoka, M., Rohani, L.P., Yamamoto, K., Wada, M., Kawabata, H., Kita, K.,

Yanase, H., Shimizu, S., 1997. Enzymatic production of ethyl (R)-4-chloro-

3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutano-

ate by an Escherichia coli transformant expressing the aldehyde reductase

gene from yeast. Appl. Microbiol. Biotechnol. 48, 699–703.

Kataoka, M., Kita, K., Wada, M., Yasohara, Y., Hasegawa, J., Shimizu, S., 2003.

Novel bioreduction system for the production of chiral alcohols. Appl.

Microbiol. Biotechnol. 62, 437–445.

Kataoka, M., Kotaka, A., Thiwthong, R., Wada, M., Nakamori, S., Shimizu, S.,

2004. Cloning and overexpression of the old yellow enzyme gene of

Candida macedoniensis, and its application to the production of a chiral

compound. J. Biotechnol. 114, 1–9.

Kaup, B., Bringer-Meyer, S., Sahm, H., 2004. Metabolic engineering of

Escherichia coli: construction of an efficient biocatalyst for D-mannitol

formation in a whole-cell biotransformation. Appl. Microbiol. Biotechnol.

64, 333–339.

Kula, M.-R., Wandrey, C., 1987. Continuous enzymatic transformation in an

enzyme-membrane reactor with simultaneous NADH regeneration. Meth-

ods Enzymol. 136, 9–21.

Labrou, N.E., Rigden, D.J., Clonis, Y.D., 2000. Characterization of the NAD+

binding site of Candida boidinii formate dehydrogenase by affinity

labelling and site-directed mutagenesis. Eur. J. Biochem. 267, 6657–

6664.

Labrou, N.E., Rigden, D.J., 2001. Active-site characterization of Candida

boidinii formate dehydrogenase. Biochem. J. 354, 455–463.

Lamzin, V.S., Dauter, Z., Popov, V.O., Harutyunyan, E.H., Wilson, K.S., 1994.

High resolution structures of holo and apo formate dehydrogenase. J. Mol.

Biol. 236, 759–785.

Lamzin, V.S., Dauter, Z., Wilson, K.S., 1995. How nature deals with stereo-

isomers. Curr. Opin. Struct. Biol. 5, 830–836.

LaReau, R.D., Anderson, V.E., 1989. Lactate dehydrogenase displays absolute

stereospecificity in the transfer of the prochiral hydrogen of NADH. J. Biol.

Chem. 264, 15338–15343.

Larsson, P., Oyston, P.C., Chain, P., Chu, M.C., Duffield, M., Fuxelius, H.H.,

Garcia, E., Halltorp, G., Johansson, D., Isherwood, K.E., Karp, P.D.,

Larsson, E., Liu, Y., Michell, S., Prior, J., Prior, R., Malfatti, S., Sjostedt,

A., Svensson, K., Thompson, N., Vergez, L., Wagg, J.K., Wren, B.W.,

Lindler, L.E., Andersson, S.G., Forsman, M., Titball, R.W., 2005. The

complete genome sequence of Francisella tularensis, the causative agent of

tularemia. Nat. Genet. 37, 153–159.

Li, L., Bannantine, J.P., Zhang, Q., Amonsin, A., May, B.J., Alt, D., Banerji, N.,

Kanjilal, S., Kapur, V., 2005. The complete genome sequence of Myco-

bacterium avium subspecies paratuberculosis. Proc. Natl. Acad. Sci. U.S.A.

102, 12344–12349.

Liese, A., Villela, M., 1999. Production of fine chemicals using biocatalysis.

Curr. Opin. Biotechnol. 10, 595–603.

Liese, A., 2005. Technical application of biological principles in asymmetric

catalysis. Adv. Biochem. Eng./Biotechnol. 92, 197–224.

Liu, Y., Xu, Z., Jing, K., Jiang, X., Lin, J., Wang, F., Cen, P., 2005. Asymmetric

reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydro-

xybutanoate with two co-existing, recombinant Escherichia coli strains.

Biotechnol. Lett. 27, 119–125.

Loftus, B.J., Fung, E., Roncaglia, P., Rowley, D., Amedeo, P., Bruno, D.,

Vamathevan, J., Miranda, M., Anderson, I.J., Fraser, J.A., Allen, J.E., Bosdet,

I.E., Brent, M.R., Chiu, R., Doering, T.L., Donlin, M.J., D’Souza, C.A., Fox,

D.S., Grinberg, V., Fu, J., Fukushima, M., Haas, B.J., Huang, J.C., Janbon, G.,

Jones, S.J., Koo, H.L., Krzywinski, M.I., Kwon-Chung, J.K., Lengeler, K.B.,

Maiti, R., Marra, M.A., Marra, R.E., Mathewson, C.A., Mitchell, T.G.,

Pertea, M., Riggs, F.R., Salzberg, S.L., Schein, J.E., Shvartsbeyn, A., Shin,

H., Shumway, M., Specht, C.A., Suh, B.B., Tenney, A., Utterback, T.R.,

Wickes, B.L., Wortman, J.R., Wye, N.H., Kronstad, J.W., Lodge, J.K.,

Heitman, J., Davis, R.W., Fraser, C.M., Hyman, R.W., 2005. The genome

of the basidiomycetous yeast and human pathogen Cryptococcus neofor-

mans. Science 307, 1321–1324.

Mathews, M.B., Vennesland, B., 1950. Enzymic oxidation of formic acid. J.

Biol. Chem. 186, 667–682.

Maurer, S.C., Schulze, H., Schmid, R.D., Urlacher, V., 2003. Immobilisation of

P450BM-3 and an NADP+ cofactor recycling system: towards a technical

application of heme-containing monooxygenases in fine chemical synthesis.

Adv. Synth. Catal. 345, 802–810.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110 109

Maurer, S.C., Kuehnel, K., Kaysser, L.A., Eiben, S., Schmid, R.D., Urlacher,

V.B., 2005. Catalytic hydroxylation in biphasic systems using CYP102A1

mutants. Adv. Synth. Catal. 347, 1090–1098.

Mesentsev, A.V., Lamzin, V.S., Tishkov, V.I., Ustinnikova, T.B., Popov, V.O.,

1997. Effect of pH on kinetic parameters of NAD+-dependent formate

dehydrogenase. Biochem. J. 321, 475–480.

Mitsunaga, T., Tanaka, Y., Yoshida, T., Watanabe, K., 2000. New Hyphomi-

crobium sp. formate dehydrogenase gene for producing formate dehydro-

genase of high specific activity, high temperature stability and high pH

stability. Patent of Japan JP245471A2, 12.09.2000.

Nagao, T., Mitamura, T., Wang, X.H., Negoro, S., Yomo, T., Urabe, I., Okada,

H., 1992. Cloning, nucleotide sequences, and enzymatic properties of

glucose dehydrogenase isozymes from Bacillus megaterium IAM1030. J.

Bacteriol. 174, 5013–5020.

Nanba, H., Takaoka, Y., Hasegawa, J., 2003a. Purification and characterization

of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus

sp. strain KNK65MA, and cloning of the gene. Biosci. Biotechnol. Bio-

chem. 67, 2145–2153.

Nanba, H., Takaoka, Y., Hasegawa, J., 2003b. Purification and characterization

of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M,

and cloning of the gene. Biosci. Biotechnol. Biochem. 67, 720–728.

Neuhauser, W., Steininger, M., Haltrich, D., Kulbe, K.D., Nidetzky, B., 1998. A

pH-controlled fed-batch process can overcome inhibition by formate in

NADH-dependent enzymatic reductions using formate dehydrogenase-cat-

alyzed coenzyme regeneration. Biotechnol. Bioeng. 60, 277–282.

Odintseva, E.R., Popova, A.S., Rojkova, A.M., Tishkov, V.I., 2002. Role of

cysteine residues in stability of bacterial formate dehydrogenase. Bull.

Moscow Univ., Ser. 2 Chem. 43, 356–359.

Olson, B.J., Skavdahl, M., Ramberg, H., Osterman, J.C., Markwell, J., 2000.

Formate dehydrogenase in Arabidopsis thaliana: characterization and

possible targeting to the chloroplast. Plant Sci. 159, 205–212.

Omura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M.,

Takahashi, Y., Horikawa, H., Nakazawa, H., Osonoe, T., Kikuchi, H., Shiba,

T., Sakaki, Y., Hattori, M., 2001. Genome sequence of an industrial

microorganism Streptomyces avermitilis: deducing the ability of producing

secondary metabolites. Proc. Natl. Acad. Sci. U.S.A. 98, 12215–12220.

Parkhill, J., Sebaihia, M., Preston, A., Murphy, L.D., Thomson, N., Harris, D.E.,

Holden, M.T.G., Churcher, C.M., Bentley, S.D., Mungall, K.L., Cerdeno-

Tarraga, A.M., Temple, L., James, K., Harris, B., Quail, M.A., Achtman, M.,

Atkin, R., Baker, S., Basham, D., Bason, N., Cherevach, I., Chillingworth,

T., Collins, M., Cronin, A., Davis, P., Doggett, J., Feltwell, T., Goble, A.,

Hamlin, N., Hauser, H., Holroyd, S., Jagels, K., Leather, S., Moule, S.,

Norberczak, H., O’Neil, S., Ormond, D., Price, C., Rabbinowitsch, E.,

Rutter, S., Sanders, M., Saunders, D., Seeger, K., Sharp, S., Simmonds, M.,

Skelton, J., Squares, R., Squares, S., Stevens, K., Unwin, L., Whitehead, S.,

Barrell, B.G., Maskell, D.J., 2003. Comparative analysis of the genome

sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella

bronchiseptica. Nat. Genet. 35, 32–40.

Popov, V.O., Shumilin, I.A., Ustinnikova, T.B., Lamzin, V.S., Egorov, Ts.A.,

1990. NAD-dependent formate dehydrogenase from methylotrophic bac-

terium Pseudomonas sp.101. I. Amino acid sequence. Bioorgan. Chem. 16,

324–335 (Russ.).

Popov, V.O., Lamzin, V.S., 1994. NAD+-dependent formate dehydrogenase.

Biochem. J. 301, 625–643.

Popov, V.O., Tishkov, V.I., 2003. NAD+-dependent formate dehydrogenase.

From a model enzyme to a versatile biocatalyst. In: Uversky, V.N. (Ed.),

Protein Structures: Kaleidoscope of Structural Properties and Functions.

Research Signpost, Kerala, pp. 441–473.

Relyea, H.A., van der Donk, W.A., 2005. Mechanism and applications of

phosphite dehydrogenase. Bioorg. Chem. 33, 171–189.

Relyea, H.A., Vrtis, J.M., Woodyer, R., Rimkus, S.A., van der Donk, W.A.,

2005. Inhibition and pH dependence of phosphite dehydrogenase. Bio-

chemistry 44, 6640–6649.

Resch, A., Rosenstein, R., Nerz, C., Gotz, F., 2005. Differential gene expression

profiling of Staphylococcus aureus cultivated under biofilm and planktonic

conditions. Appl. Environ. Microbiol. 71, 2663–2676.

Rissom, S., Schwarz-Linek, U., Vogel, M., Tishkov, V.I., Kragl, U., 1997.

Synthesis of chiral epsilon-lactones in a two-enzyme system of cyclohex-

anone mono-oxygenase and formate dehydrogenase with integrated bubble-

free aeration. Tetrahedron-Asymmetry 8, 2523–2526.

Rojkova, A.M., Galkin, A.G., Kulakova, L.B., Serov, A.E., Savitsky, P.A.,

Fedorchuk, V.V., Tishkov, V.I., 1999. Bacterial formate dehydrogenase.

Increasing the enzyme thermal stability by hydrophobization of alpha

helices. FEBS Lett. 445, 183–188.

Rozzell, J.D., Hua, L., Mayhew, M., Novick, S., 2004. Mutants of enzymes and

methods for their use. US Patent Application Publication US2004/0115691,

17.06.2004.

Sadykhov, E.G., Serov, A.E., Voinova, N.S., Uglanova, S.V., Petrov, A.S.,

Alexeeva, A.A., Kleimenov, S.Yu., Popov, V.O., Tishkov, V.I., 2006.

Comparable study of thermal stability of formate dehydrogenases from

microorganisms and plants. Appl. Biochem. Microbiol. 42, N3.

Sakai, Y., Murdanoto, A.P., Konishi, T., Iwamatsu, A., Kato, N., 1997.

Regulation of the formate dehydrogenase gene, FDH1, in the methylo-

trophic yeast Candida boidinii and growth characteristics of an FDH1-

disrupted strain on methanol, methylamine, and choline. J. Bacteriol. 179,

4480–4485.

Saleeba, J.A., Cobbett, C.S., Hynes, M.J., 1992. Characterization of the amdA-

regulated aciA gene of Aspergillus nidulans. Mol. Gen. Genet. 235, 349–

358.

San, K.Y., Bennett, G.N., Berrios-Rivera, S.J., Vadali, R.V., Yang, Y.T., Horton,

E., Rudolph, F.B., Sariyar, B., Blackwood, K., 2002. Metabolic engineering

through cofactor manipulation and its effects on metabolic flux redistribu-

tion in Escherichia coli. Metab. Eng. 4, 182–192.

Sanchez, A.M., Bennett, G.N., San, K.Y., 2005. Effect of different levels of

NADH availability on metabolic fluxes of Escherichia coli chemostat

cultures in defined medium. J. Biotechnol. 117, 395–405.

Schwarz-Linek, U., Krodel, A., Ludwig, F.A., Schulze, A., Rissom, S., Kragl,

U., Tishkov, V.I., Vogel, M., 2001. Synthesis of natural product precursors

by Baeyer-Villiger oxidation with cyclohexanone monooxygenase from

Acinetobacter. Synthesis-Stuttgart 33, 947–951.

Seelbach, K., Riebel, B., Hummel, W., Kula, M.-R., Tishkov, V.I., Egorov,

A.M., Wandrey, C., Kragl, U., 1996. A novel, efficient regenerating method

of NADPH using a new formate dehydrogenase. Tetrahedron Lett. 37,

1377–1380.

Serov, A.E., Popova, A.S., Fedorchuk, V.V., Tishkov, V.I., 2002. Engineering of

coenzyme specificity of formate dehydrogenase from Saccharomyces cer-

evisiae. Biochem. J. 367, 841–847.

Serov, A.E., 2002. Structure–function relationship in recombinant formate

dehydrogenases from baker’s yeast and methylotrophic bacterium. Ph.D.

Thesis. M.V. Lomonosov Moscow State University, Moscow, Russian

Federation.

Serov, A.E., Tishkov, V.I., 2002. Role of Pro residues in stability of prokaryotic

and euacaryotic formate dehydrogenases. Bull. Moscow Univ., Ser. 2 Chem.

43, 345–349.

Serov, A.E., Odintzeva, E.R., Uporov, I.V., Tishkov, V.I., 2005. Use of Rama-

chandran plot for increasing thermal stability of bacterial formate dehy-

drogenase. Biochemistry (Mosc.) 70, 804–808.

Shaked, Z., Whitesides, G.M., 1980. Enzyme-catalyzed organic synthesis:

NADH regeneration by using FDH. J. Am. Chem. Soc. 102, 7104–

7105.

Shinoda, T., Satoh, T., Mineki, S., Iida, M., Taguchi, H., 2002. Cloning,

nucleotide sequencing, and expression in Escherichia coli of the gene

for formate dehydrogenase of Paracoccus sp.12-A, a formate-assimilating

bacterium. Biosci. Biotechnol. Biochem. 66, 271–276.

Shinoda, T., Arai, K., Shigematsu-Iida, M., Ishikura, Y., Tanaka, S., Yamada, T.,

Kimber, M.S., Pai, E.F., Fushinobu, S., Taguchi, H., 2005. Distinct con-

formation-mediated functions of an active site loop in the catalytic reactions

of NAD-dependent D-lactate dehydrogenase and formate dehydrogenase. J.

Biol. Chem. 280, 17068–17075.

Schirwitz, K., Schmidt, A., Lamzin, V.S., 2005. Site-directed mutagenesis and

high-resolution structures of Candida boidinii formate dehydrogenase.

HASYLAB Annual Report, DESY, Hamburg. http://www-hasylab.desy.de/

science/annual_reports/2005_report/part2/contrib/72/14698.pdf.

Slusarczyk, H., Felber, S., Kula, M.R., Pohl, M., 2000. Stabilization of NAD-

dependent formate dehydrogenase from Candida boidinii by site-directed

mutagenesis of cysteine residues. Eur. J. Biochem. 267, 1280–1289.

V.I. Tishkov, V.O. Popov / Biomolecular Engineering 23 (2006) 89–110110

Slusarczyk, H., Felber, S., Kula, M.-R., Pohl, M., 2003. Novel mutants of

formate dehydrogenase from Candida boidinii. US Patent Application

Publication US2003/0157664, 21.09.2003.

Tishkov, V.I., Galkin, A.G., Egorov, A.M., 1991. NAD-dependent formate

dehydrogenase of methylotrophic bacteria Pseudomonas sp.101: cloning,

expression, and study of the gene structure. Dokl. Akad. Nauk SSSR 317,

745–748.

Tishkov, V.I., Galkin, A.G., Marchenko, G.N., Egorova, O.A., Sheluho, D.V.,

Kulakova, L.B., Dementieva, L.A., Egorov, A.M., 1993. Catalytic proper-

ties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants

containing Cys-255-Ser and Cys-255-Met replacements. Biochem. Bio-

phys. Res. Commun. 192, 976–981.

Tishkov, V.I., Galkin, A.G., Fedorchuk, V.V., Savitsky, P.A., Rojkova, A.M.,

Gieren, H., Kula, M.-R., 1999. Pilot scale production and isolation of

recombinant NAD+- and NADP+-specific formate dehydrogenases. Bio-

technol. Bioeng. 64, 187–193.

Tishkov, V.I., Popov, V.O., 2004. Catalytic mechanism and application of

formate dehydrogenase. Biochemistry (Mosc.) 69, 1252–1267.

Urlacher, V.B., Schmid, R.D., 2004. Protein engineering of the cytochrome

P450 monooxygenase from Bacillus megaterium. Methods Enzymol. 388,

208–224.

Urlacher, V.B., Makhsumkhanov, A., Schmid, R.D., 2005. Biotransformation of

beta-ionone by engineered cytochrome P450 BM-3. Appl. Microbiol.

Biotechnol Published online: July 7, 2005.

van der Donk, W.A., Zhao, H.M., 2003. Recent developments in pyridine

nucleotide regeneration. Curr. Opin. Biotechnol. 14, 421–426.

Vinals, C., Depiereux, E., Feytmans, E., 1993. Prediction of structurally

conserved regions of D-specific hydroxy acid dehydrogenases by multiple

alignment with formate dehydrogenase. Biochem. Biophys. Res. Commun.

192, 182–188.

Vrtis, J.M., White, A.K., Metcalf, W.W., van der Donk, W.A., 2005. Phosphite

dehydrogenase: versatile cofactor-regeneration enzyme. Angew. Chem. Int.

Ed. Engl. 41, 3257–3259.

Wada, M., Yoshizumi, A., Noda, Y., Kataoka, M., Shimizu, S., Takagi, H.,

Nakamori, S., 2003. Production of a doubly chiral compound (4R,6R)-4-

hydroxy-2,2,6-trimethylcyclohexanone, by two-step enzymatic asymmetric

reduction. Appl. Environ. Microbiol. 69, 933–937.

Weerasinghe, P.A., Weerasekera, M.L.M.C., van Holm, L.H.J., 1999. Use of

isoenzymes to differentiate growth categories of Pericopsis mooniana trees.

Biol. Plant. 42, 41–547.

Weinhold, E.G., Glasfeld, A., Ellington, A.D., Benner, S.A., 1991. Structural

determinants of stereospecificity in yeast alcohol dehydrogenase. Proc.

Natl. Acad. Sci. U.S.A. 88, 8420–8424.

Weuster-Botz, D., Paschold, H., Striegel, B., Gieren, H., Kula, M.-R., Wandrey,

C., 1994. Continuous computer controlled production of formate dehydro-

genase (FDH) and isolation on a pilot scale. Chem. Eng. Technol. 17, 131–

137.

Wichmann, R., Wandrey, C., Buckmann, A.F., Kula, M.R., 1981. Continuous

enzymatic transformation in an enzyme membrane reactor with simu-

ltaneous NAD(H) regeneration. Biotechnol. Bioeng. 23, 2789–

2802.

Wichmann, R., Vasic-Racki, D., 2005. Cofactor regeneration at the lab scale.

Adv. Biochem. Eng./Biotechnol. 92, 225–260.

Woodyer, R., van der Donk, W.A., Zhao, H., 2003. Relaxing the nicotinamide

cofactor specificity of phosphite dehydrogenase by rational design. Bio-

chemistry 42, 11604–11614.

Woodyer, R., Zhao, H., van der Donk, W.A., 2005. Mechanistic investigation of

a highly active phosphite dehydrogenase mutant and its application for

NADPH regeneration. FEBS J. 72, 3816–3827.

Xu, Z., Liu, Y., Fang, L., Jiang, X., Jing, K., Cen, P., 2005. Construction of a

two-strain system for asymmetric reduction of ethyl 4-chloro-3-oxobutano-

ate to (S)-4-chloro-3-hydroxybutanoate ethyl ester. Appl. Microbiol. Bio-

technol. Published on-line: September 21, 2005.

Yamamoto, H., Mitsuhashi, K., Kimoto, N., Kobayashi, Y., Esaki, N., 2005.

Robust NADH-regenerator: improved alpha-haloketone-resistant formate

dehydrogenase. Appl. Microbiol. Biotechnol. 67, 33–39.

Yun, H., Choi, H.L., Fadnavis, N.W., Kim, B.G., 2005. Stereospecific

synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli

BL21 overexpressing YiaE from Escherichia coli K12 and glucose

dehydrogenase from Bacillus subtilis. Biotechnol. Prog. 21, 366–371.