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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: [email protected], [email protected] (V.I. Tishkov), [email protected] (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. Thisprovides 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 scaleenzyme production with a comparatively low production
cost.
(5) B
acterial and yeast FDHs are sufficiently stable to be usedin 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 kineticproperties;
- 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 inEscherichia 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
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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
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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
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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).
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