Purification and characterization of an α-haloketone-resistant formate dehydrogenase from Thiobacillus sp. strain KNK65MA, and cloning of the gene

Article abstract of DOI:10.1271/bbb.67.2145

Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to α-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with α-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent K_m values for formate and NAD⁺ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.

Full text of DOI:10.1271/bbb.67.2145

This article was downloaded by: [UAA/APU Consortium Library]
On: 14 October 2014, At: 15:05
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Purification and Characterization of an α-
Haloketone-resistant Formate Dehydrogenase from
Thiobacillus sp. Strain KNK65MA, and Cloning of the
Gene
Hirokazu NANBAa, Yasuko TAKAOKAa & Junzo HASEGAWAa
a Fine Chemicals Research Laboratories, Kaneka Corporation1-8 Miyamae-machi,
Takasago-cho, Takasago, Hyogo 676-8688, Japan
Published online: 22 May 2014.
To cite this article: Hirokazu NANBA, Yasuko TAKAOKA & Junzo HASEGAWA (2003) Purification and Characterization of
an α-Haloketone-resistant Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA, and Cloning of the Gene,
Bioscience, Biotechnology, and Biochemistry, 67:10, 2145-2153, DOI: 10.1271/bbb.67.2145
To link to this article: http://dx.doi.org/10.1271/bbb.67.2145
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Biosci. Biotechnol. Biochem., 67 (10), 2145–2153, 2003
Puriˆcation and Characterization of an a-Haloketone-resistant Formate
Dehydrogenase from Thiobacillus sp. Strain KNK65MA, and Cloning
of the Gene
Hirokazu NANBA,† Yasuko TAKAOKA, and Junzo HASEGAWA
Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho,
Takasago, Hyogo 676-8688, Japan
Received April 10, 2003; Accepted July 7, 2003
Thiobacillus sp. strain KNK65MA, which produced
an NAD-dependent formate dehydrogenase (FDH)
NADP(H), it is typically coupled to the reaction that
regenerates the cofactor.4–8) Therefore, the enzyme
used for the regeneration must also be stable and
highly resistant to
i.e., the enzyme showed no loss of activity after a 5-h
incubation with -haloketones, such as ethyl 4-chloro-
a-haloketones, was newly isolated,
resistant to a-haloketones.
a
The use of NAD-dependent FDH,8–24) which cata-
lyzes the oxidation of formate to carbon dioxide with
the reduction of NAD＋ to NADH, is suitable for
practical use in the regeneration of the cofactor,
because no by-products from this regeneration reac-
tion accumulate in the reaction mixture; therefore,
the product is easily isolated, the amount of waste is
very low, and the thermodynamic equilibrium of the
reaction is favorable. Various NAD-dependent
FDHs from plants,10,11) methylotrophic yeasts,12–16)
and bacteria17–24) have been investigated. Among
3-oxobutanoate. The enzyme was also resistant to SH
reagents. The enzyme, puriˆed to homogeneity, was a
dimer composed of identical subunits. The speciˆc
activity was 7.6 u mg, and the apparent Km values for
W
formate and NAD＋ were 1.6 and 0.048 m
, respec-
M
tively. The cloned gene of FDH contained one open
reading frame (ORF) of 1206 base pairs, predicted to
encode a polypeptide of 401 amino acids, with a calcu-
lated molecular weight of 44021; this gene was highly
expressed in E. coli cells. The deduced amino acid
sequence of this FDH had high identity to other bacteri-
al FDHs.
them, FDHs from Candida, ,
25) Pseudomonas 26,27) and
Mycobacterium24) have been improved, i.e., their
resistance to oxidation or thermal inactivation has
been increased by site-directed mutagenesis, as en-
zyme stability is known to be important for practical
use. However, no reports have referred to the stabil-
Key words: NAD-dependent formate dehydrogenase;
Thiobacillus; a-haloketone-resistance
Optically active compounds have been widely
recognized as important synthetic intermediates
ity of these FDHs in the presence of
To obtain FDHs suitable for the economical
production of chiral -halohydrins, we attempted to
discover novel FDHs that might show high resistance
to -haloketones. In this report, we discuss the puriˆ-
a-haloketones.
for pharmaceuticals.1) Among them, chiral
a
-halo-
a
hydrins have been recognized as promising building
blocks for asymmetric organic synthesis, i.e., alkyl
a
(
S
)-4-halo-3-hydroxybutanoate and alkyl
(
R
)-4-
cation and characterization of NAD-dependent FDH
derived from newly isolated Thiobacillus sp. strain
KNK65MA. We also discuss the cloning and expres-
sion of the FDH gene in E. coli. In this context, the
halo-3-hydroxybutanoate have been used as synthetic
intermediates of various inhibitors of HMG-CoA
reductase2) and
L
-carnitine synthesis,3) respectively.
For the preparation of these chemicals, enzymatic
issue of resistance to a-haloketones is also addressed.
reduction has proven to be a useful tool.4) Although
enzymatic reduction required
a-haloketones as
Materials and Methods
substrates, these ketones have high reactivity and
they inactivate enzymes during this type of reaction.
Therefore, it is considered as advantageous if en-
zymes with high resistance to such substrates can be
used. On the other hand, as the enzymatic reduction
requires expensive cofactors such as NAD(H) or
Chemicals. All chemicals used in this study were
the best available commercial products.
Media and culture conditions. For the screening of
microorganisms with FDH activity, medium A,
†
To whom correspondence should be addressed. Fax:
81-794-45-2668; E-mail: Hirokazu.Nanba kaneka.co.jp
＋ ＠
Abbreviations: FDH, formate dehydrogenase; ORF, open reading frame; PCR, polymerase chain reaction; IPCR, inverted PCR; SDS-
PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

2146
H. NANBA et al
.
comprised of 6.7 g of Bacto yeast nitrogen base
without amino acids (Difco), 0.1 g bonito ˆllet
extract-type CR (Riken Vitamin), 0.1 g yeast extract
(Difco), 0.1 g polypeptone (Nippon Seiyaku), 4 g
K2HPO4, and 8 g methanol, in 1 liter of tap
water (pH 9.0), was used. Thiobacillus sp. strain
KNK65MA was inoculated into 7 ml of medium B,
comprised of 10 g glycerol, 10 g tryptone (Difco), 5 g
yeast extract, 5 g NaCl, and 20 g methanol, in 1 liter
of tap water (pH 7.0), in a test tube, was cultured at
5 min at 309C, and then assayed. From the ratio of
the activity before and after the incubation with
ethyl 4-chloro-3-oxobutanoate, the microorganisms
producing FDH with high resistance to ethyl 4-chlo-
ro-3-oxobutanoate were selected.
Puriˆcation of FDH. All procedures were done at
9
4 C, and all buŠers used were potassium phosphate
buŠer containing 1 m
M
dithiothreitol and 1 m
M
EDTA. Step 1: Washed strain KNK65MA cells,
collected from 3600 ml of the cultured broth, were
289C for 2 days with shaking, inoculated into 100 ml
of medium A containing 20 g per liter of methanol in
a 500-ml shaking ‰ask, and then the mixtures were
suspended in 180 ml of 100 m
M
buŠer (pH 7.0); the
cells were then disrupted by sonication. Step 2: The
enzyme solution was fractionated with ammonium
cultured at 28
9C for 7 days with shaking. E. coli
HB101 carrying pFT002 was cultured in 10 ml of
sulfate (25–60
was dissolved in 10 m
Step 3: The enzyme solution was placed on a DEAE-
z
saturation), and then the precipitate
2YT medium28) in a test tube at 30
9
C for 25 h with
M
buŠer (pH 6.5) and dialyzed.
shaking. The recombinant E. coli cells were harvest-
×
ed from 1 ml of cultured broth, suspended in 1 ml of
Sepharose column (4.5 14 cm), and then eluted
with 10 m buŠer (pH 6.5) and 100 m buŠer
(pH 6.5). Step 4: Ammonium sulfate was added to
the active fractions (24 saturation), and then the
solution was placed on a phenyl-Toyopearl 650M
0.1
M
potassium phosphate buŠer (pH 7), disrupted
M
M
by sonication, centrifuged to remove the cell debris,
and then assayed. E. coli HB101 carrying pFA002,
which contained the FDH gene from Ancylobacter
aquaticus strain KNK607M, was cultured as de-
scribed previously.17) Candida boidinii ATCC32195
was inoculated into medium C, comprised of 6.7 g
Bacto yeast nitrogen base without amino acids, 1 g
bonito ˆllet extract-type CR, 1 g yeast extract, 1 g
polypeptone, 2 g KH2PO4, 2 g K2HPO4, and 8 g
methanol, in 1 liter of tap water (pH 6.0), and then
z
×
(Tosoh) column (2.5 20 cm), eluted with 100 m
buŠer (pH 6.0) containing 24 -saturated ammoni-
um sulfate, and then eluted with a reduction in
ammonium sulfate concentration to 0 saturation.
M
z
z
The combined active fractions were concentrated by
ammonium sulfate, and then the precipitate was
dissolved in 10 m buŠer (pH 6.5) and dialyzed. Step
M
the mixture was cultured at 28
shaking.
9
C for 72 h with
5: The enzyme solution was placed on a Blue
Sepharose 6 Fast Flow (Pharmacia Biotech) column
×
M
(1.5 13 cm), eluted with 10 m buŠer (pH 6.5), and
Screening methods. The ˆrst screening for the
isolation of microorganisms with FDH activity was
done as follows. A spoonful of soil was suspended in
then with an increase in the NaCl concentration to
0.5 . This operation was done twice, and then
M
the eluted active fractions were concentrated by
ultraˆltration. Step 6: The enzyme solution was
1 ml of saline, and then 70 ml of the supernatant was
used to inoculate 7 ml of medium A in a test tube,
which contained methanol as the sole carbon source.
placed on
a
Gigapite (Seikagaku Corporation)
6.5 cm), and then eluted with buŠer
column (1.5
×
After aerobic cultivation at 30
9C until growth was
(pH 6.5); this procedure was increased stepwise in
detected visually, growth was measured by the absor-
bance at 600 nm. Then, cells from 1.5 ml of cultured
broth were harvested, suspended in 0.5 ml of sub-
potassium phosphate concentration (2 m
M
, 50 m ,
M
70 m , and 200 m ). This operation was done twice,
M
M
and then the eluted active fractions were collected
and concentrated to 6.0 ml by ultraˆltration.
strate solution comprised of 100 m
phate buŠer, 500 m sodium formate, 1 m
and 1 Triton X-100 (pH 7.0), and were incubated
at 30 C for 20 h with shaking. The NADH formed by
M
potassium phos-
M
M
NAD＋,
z
9
Measurement of FDH activity. The standard FDH
assay was done as follows. The assay mixture, com-
this method was measured by the increase of absor-
bance at 340 nm. The cultured broths expressing a
high ratio of absorbance at 340 nm per 600 nm were
selected. For the second screening, the selected
broths were inoculated into medium A, cultured un-
der the same conditions as those described above,
and cells from 5 ml of cultured broth were harvested,
prised of 1.25 ml of 100 m
buŠer (pH 7.0), 1.5 ml of 1
100 m potassium phosphate buŠer (pH 7.0), 150
of 100 m l of enzyme solution,
NAD＋, and 100
was incubated at 30 C, and the increase in absor-
M
potassium phosphate
M
sodium formate in
M
ml
M
m
9
bance at 340 nm was monitored. One unit of FDH
was deˆned as the amount that catalyzed the reduc-
suspended in 0.5 ml of 100 m
M
potassium phosphate
tion of 1
The m and Vmax of the puriˆed enzyme were calcu-
m
mol of NAD＋ per min.
buŠer (pH 7.0), disrupted by sonication, centrifuged
to remove the cell debris, and then assayed. The cell-
free extracts were mixed with equal amounts of
K
lated from double-reciprocal Lineweaver-Burk plots.
As regards the formate, a standard assay mixture
containing various concentrations of formate and
20 m
M
ethyl 4-chloro-3-oxobutanoate, incubated for

Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA
2147
Table 1. Oligonucleotides Used as Primers for PCR
Primer Sequence
-GCN AAR ATN CTN TGY GT-3
PCR using the genomic DNA as a template with SP1
and AP1 primers. The reaction mixture was com-
posed of each primer, the genomic DNA, the four
deoxynucleotides, ExTaq DNA polymerase, and 10x-
buŠer for ExTaq (Takara).
For the Southern hybridization, genomic DNA
(500 ng) digested with each restriction enzyme was
transferred onto a Hybond-N＋ ˆlter (Amersham
Pharmacia Biotech), as described in the manufac-
turer's instructions, and then hybridization was done
with a probe that was a segment of the FDH gene
ampliˆed by PCR using the genomic DNA as a tem-
plate with SP1 and AP1 primers. Hybridization,
preparation of the probe, and detection of the signals
were done according to the manufacturer's instruc-
tions included with the Gene Images kit (Amersham
Pharmacia Biotech).
SP1
AP1
SP2
AP2
SP3
AP3
SP4
AP4
5
?
?
?
5
5
-GGY TGN GGR AAC CAN ACR TC-3
-GAG ACG CTG AAG CTG TTC AA-3
-TAG TGG TCG ATC TTC GGC AGA TCG T-3
-ACA CTG CAG ATA CGC GCG AGA GGA GAT-3
-TCT GAA TTC GTT CGC CTG GTC GGT CTG T-3
-ACG CAT ATG GCG AAA ATA CTT TGC-3
-AGT CTG CAG TTA GCC GGC CTT CTT GAA-3?
?
?
?
5
?
?
5
?
?
?
5
?
5
?
?
5?
N is indicated as A or G or C or T; R is indicated as A or G; Y is indicated
as C or T.
The underlined portions indicate the recognition site of restriction
enzymes; Pst I site for SP3, EcoRI site for AP3, NdeI site for SP4, and Pst
site for AP4.
I
5 m
containing various concentrations of NAD＋ and
500 m formate was used.
M
NAD＋ was used; for the NAD＋, a mixture
According to the method of Triglia et al.,31) IPCR
was done as follows. The genomic DNA was digested
with EcoRI, and then self-ligated with T4 DNA
ligase. PCR was done using the self-ligated DNA as a
template with SP2 and AP2 primers. The reaction
mixture was composed of each primer, the self-
ligated DNA solution, the four deoxynucleotides,
LATaq DNA polymerase, and 2x-GC buŠer I for
LATaq (Takara). The ampliˆed fragments were used
directly for the nucleotide sequencing.
M
Analytical methods. The protein concentration
29)
was measured by the method of Lowry et al
.
using
bovine serum albumin as a standard. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) was done using 10
z
polyacrylamide gels
30)
and the methods of Laemmli et al
.
The gel was
stained with Coomassie brilliant blue R250. High-
performance gel permeation chromatography on a
G3000SWXL column (Tosoh) was done to measure
the molecular mass of the native enzyme using the
protein molecular weight standards kit MS II
A fragment containing the ORF of the FDH gene
was ampliˆed by PCR using the genomic DNA as a
template with the SP3 and AP3 primers. The reaction
mixture was composed of each primer, the genomic
DNA, the four deoxynucleotides, LATaq DNA poly-
merase, and 2x-GC buŠer I for LATaq (Takara). A
pFT001 was constructed by cloning the ampliˆed
fragment into the Pst I-EcoRI site of pUC19. The
same manipulations, from the ampliˆcation to the
cloning into pUC19, were done three times, and all of
the sequences of the cloned fragments were analyzed.
(Boehringer Ingelheim). The
N-terminal amino acid
sequence of the puriˆed FDH was analyzed by
automated Edman degradation using a model 49X
Procise (Applied Biosystems).
Oligonucleotides used as primers for PCR.
Sequences of the primers for polymerase chain reac-
tion (PCR) are shown in Table 1. Synthetic
oligonucleotides were designed as degenerated
All of the DNA manipulations were done using stan-
28)
dard methods, as described by Sambrook et al
.
primers; SP1 corresponding to the
N-terminal amino
acid sequence AKILCV, and AP1 corresponding to
the amino acid sequence DVWFPQP, conserved
Construction of an expression vector. To e‹cient-
ly express the FDH gene in E. coli, a plasmid,
pFT002, was constructed. PCR was done using
genomic DNA as a template with SP4 and AP4
primers. The ampliˆed fragment was subcloned
between NdeI and PstI sites in pUCNT, which was
constructed as an expression vector by Nanba et
al.,33) and sequenced. The resultant plasmid, pFT002,
was introduced into E. coli HB101.
among FDHs of Aspergillus
ula
, Neurospora, Hansen-
,
Mycobacterium, and Pseudomonas. For invert-
ed PCR (IPCR),31) SP2 and AP2 primers were used,
whereby both SP2 and AP2 corresponded to the
partial sequence in the fragment ampliˆed by PCR
with the genomic DNA as a template and the de-
generated SP1 and AP1 primers. SP3 and AP3
primers corresponded to the partial sequence in the
fragment ampliˆed by IPCR. SP4 and AP4 primers
DNA sequencing. The nucleotide sequences were
analyzed by the dideoxy chain termination method34)
with an ABI PRISM 310 Genetic Analyzer (Applied
Biosystems), using a DNA Sequencing kit, BigDye
Terminator V2.5 Cycle Sequencing Ready Reaction
(Applied Biosystems). Similarity searches for the
deduced amino acid sequence were done with the
corresponded to the deduced
terminal region of the FDH, respectively.
N-terminal and C-
Gene cloning. The genomic DNA was prepared
from strain KNK65MA by the miniprep method.32)
0.95 kbp portion of the FDH gene was ampliˆed by
A

2148
H. NANBA et al
.
BLAST program of the National Center for
Biotechnology Information (NCBI) at the National
Library of Medicine. The nucleotide sequence data
has been submitted to the DDBJ, EMBL, and
GenBank nucleotide sequence databases; the data
has been assigned the accession number AB106890.
ethyl 4-chloro-3-oxobutanoate were isolated from a
total of 507 soil samples after enrichment culture
with methanol as the sole carbon source. Among
these strains, KNK65MA had the highest resistance
to ethyl 4-chloro-3-oxobutanoate, and no loss in
FDH activity was observed under the conditions of
the second screening described in Materials and
Methods. The strain expressed 0.41 units of FDH
activity per ml of the culture broth, and was identi-
ˆed as Thiobacillus sp., the FDH of which has been
reported previously.35–39)
Evaluation of resistance to a-haloketones. Cells of
E. coli HB101 (pFT002), E. coli HB101 (pFA002),
and Candida boidinii ATCC32195 were harvested,
suspended in 100 m
M
potassium phosphate buŠer
(pH 6.5; one-half volume of the cultured E. coli
broth, and a one-tenth volume of Candida), disrupt-
ed by sonication for E. coli cells, and by glass bead
for the Candida cells; the suspensions were then
centrifuged to remove the cell debris. The cell-free
extracts were incubated in the presence of the
haloketones shown in Table 3, and then assayed.
Puriˆcation and properties of FDH
The FDH was puriˆed about 26-fold (Table 2).
The puriˆed enzyme was a single protein band of
44 kDa on SDS-PAGE. The relative molecular mass
of the enzyme was estimated to be about 90 kDa,
based on gel permeation chromatography. These
results demonstrated that the enzyme is composed of
two identical subunits.
a-
Results and Discussion
The enzyme was most active at pH 5.6, and
showed relatively high activity within a wide pH
range (Fig. 1(A)). The activity was measured at vari-
ous temperatures, as shown in Fig. 1(B), and reached
Isolation of microorganisms that produce FDH
with high resistance to a-haloketones
The strains producing FDH with high resistance to
9
a maximum at about 58 C. The stability of the FDH
rendered it suitable for practical use, as the enzyme
was stable within a wide pH range (Fig. 2(A)). Fur-
Table 2. Puriˆcation of FDH from Thiobacillus sp. Strain
KNK65MA
thermore, it was stable at 50
The apparent Km values for formate and NAD＋
were 1.6 and 0.048 m , respectively, which were
lower than those of the other FDHs, with the excep-
tion of the respective Paracoccus FDH (0.036 m
for NAD＋ )21) and Pseudomonas oxalaticus FDH
(0.14 m m values
for formate) values.18) The lower
9
C or lower (Fig. 2(B)).
Total
Total
Speciˆc
Activity
Yield
Step
Activity Protein
(
z
)
M
(units)
(mg)
(units mg)
W
Crude extract
1040
895
673
166
107
63.4
3570
2040
323
30.7
15.9
8.42
0.291
0.439
2.08
5.41
6.73
7.53
100
M
Ammonium sulfate
DEAE-Sepharose
Phenyl-Toyopearl
Blue-Sepharose
Gigapite
86.1
64.7
16.0
10.3
M
K
of the enzyme were favorable for the cofactor
regeneration system because of the higher activity of
6.10
Fig. 1. EŠects of pH (A) and Temperature (B) on the Activity of FDH.
(A) Enzyme activity was assayed under the standard assay conditions using 0.063 units of puriˆed FDH, with the exception that the
following buŠers were used at 100 m : potassium phosphate (pH 5–8), reaction mixtures (pH 4.8–7.4), ( ); Tris-HCl (pH 7.5–9), reac-
M


#
tion mixtures (pH 7.4–8.9), ( ); carbonate (pH 9–11.5), reaction mixtures (pH 8.4–10.9), ( ). The relative activity was the percentage
of the activity at pH 5.6. (B) Assays were done at various temperatures for 5 min under standard assay conditions using 0.013 units of
puriˆed FDH. The relative activity was the percentage of the activity at 309C.

Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA
2149
Fig. 2. EŠects of pH (A) and Temperature (B) on the Stability of the FDH.
(A) 0.16 units of FDH were incubated at 30 C for 22 h in 0.25 ml of the following buŠers: 50 m
100 m of potassium phosphate, pH 5.3–7.9 (
9

M
of sodium acetate, pH 4.0–5.6 (

);
#
$
M
); 100 m
M
of Tris-HCl, pH 7.5–9.0 ( ); 100 m
M
of carbonate, pH 9.0–10.5 ( ). After
incubation, the remaining activity was assayed under the standard assay conditions, and was expressed as the percentage of the activity
before the incubation. (B) 0.082 units of FDH were incubated at diŠerent temperatures for 10 min in 130 l of 100 m potassium phos-
phate, pH 7.0, and then the remaining activity was assayed under the standard assay conditions, and was expressed as the percentage of
the activity after the incubation at 0 C.
m
M
9
the enzyme, which was observed even at lower
concentrations of the substrate.
Cloning and sequencing of the gene for FDH, and
comparison of the deduced amino acid sequence with
those of other FDHs
Using a variety of carboxylic acids, aldehydes, and
alcohols as substrates, the activity of the enzyme was
examined. Reduction of NAD＋ to NADH was
detected only when formate was used as a substrate
for concomitant oxidation. The following com-
pounds were judged to be inactive as substrates
because no formation of NADH was detected:
methanol, formaldehyde, ethanol, acetaldehyde,
acetic acid, glyoxylic acid, 1-propanol, propionalde-
hyde, propionic acid, and glycerol. The enzyme
was speciˆc to formate, the same as the other
FDHs.12,13,15,19,20) Some electron acceptors were also
investigated as cofactors, with formate as an electron
donor. The enzyme was most active in the presence
of NAD＋, and the activity in the presence of NADP＋
Southern hybridization was done using a portion
of the FDH gene as a probe, and the genomic DNA
digested by EcoRI gave a single band of about
4.5 kbp upon agarose gel electrophoresis. Based on
the results, IPCR was done using the genomic DNA
self-ligated at the EcoRI site as a template. Using
primers corresponding to the outside of the ORF,
and using the genomic DNA as a template, PCR was
done; then, a fragment containing the ORF of the
FDH gene was ampliˆed. A pFT001 was constructed
by insertion of 1275 bp of the ampliˆed Pst I-EcoRI
fragment into pUC19, and then the nucleotide se-
quence of the insert was analyzed. The same steps
were done three times, and all sequences of the
obtained clones were identical.
was 4.2
z
of that in the presence of NAD＋.
However, the following compounds were not
reduced: ferricyanide, nitro blue tetrazolium, phena-
zine methosulfate, benzyl viologen, and 2,6-
dichloroindophenol.
From the DNA sequence submitted to the nucleo-
tide sequence database, the ORF consisted of 1206
bases with a starting triplet, ATG, at position 32 and
an ending triplet, TGA, at position 1235. The ORF
was predicted to encode a polypeptide of 401 amino
acids, with a calculated molecular weight of 44,021.
Enzyme activity was assayed under the standard
conditions in the presence of various compounds.
＋
The enzyme was inhibited by NaN3 (1 m
M
) and Hg2
The deduced N-terminal amino acid sequence coin-
(1 m
by
(0.2 m
tively.
M
) with 100
z
inhibition, and slightly inhibited
cided with the sequence of the puriˆed FDH from
strain KNK65MA (30 amino acids, not including the
ˆfth amino acid, were identiˆed.), except for the exis-
＋
p
-chloromercuribenzoic acid (0.2 m
M
) and Cu2
inhibition, respec-
) and the metal chela-
-phenanthroline (0.2 m ),
were not associated with any inhibition of enzyme
activity. Resistance to SH reagents such as
chloromercuribenzoic acid and -ethyl maleimide
M
) with 10.7
-ethyl maleimide (1 m
) and
z and 13.6z
N
M
tence of an N-terminal methionine. The alignment of
tors, EDTA (1 m
M
o
M
the deduced amino acid sequence of this FDH with
those of other FDHs is shown in Fig. 3. The FDH of
strain KNK65MA had high identity with the other
known bacterial FDHs. The identities with the
p
-
N
17)
19,22)
was one of the characteristic properties of this FDH,
compared to Ancylobacter FDH, which lacked this
property.17)
enzymes from Ancylobacter
,
Pseudomonas
and Paracoccus21,23)
, 84.8 , and 82.5
,
24)
20)
Mycobacterium
,
Moraxella
, 91.5
,
were 89.8 , 92.0
z
z
z
z
z
,

2150
H. NANBA et al.
Fig. 3. Alignment of the Amino Acid Sequences of Various FDHs.
1, FDH of Thiobacillus sp. strain KNK65MA; 2, FDH of Ancylobacter aquaticus strain KNK607M;17) 3, FDH of Pseudomonas, sp.
101;19,22) 4, FDH of Mycobacterium vaccae N10;24) 5, FDH of Moraxella, sp.;20) 6, FDH of Paracoccus sp. 12-A;21,23) 7, FDH of Candida
boidinii; ; .
25) 8, FDH-like protein of Aspergillus nidulans 40) 9, FDH of Solanum tuberosum 11) The conserved amino acids are marked in
black.
respectively. Enzymes from eucaryotes, such as those
from yeast (Candida boidinii),25) fungi (Aspergillus
nidulans),40) and plants (potato, Solanum tubero-
to an approximately 28-fold increase over the activity
of strain KNK65MA.
sum),11) showed lower identities (49.4
z, 53.0
z, and
Resistance to a-haloketones
51.7
z, respectively). Using the expression plasmid
As shown in Table 3, the FDH of strain
pFT002, the FDH gene was e‹ciently expressed. The
activity of E. coli HB101 carrying pFT002 was 11.3
units per ml of the cultured broth, which amounted
KNK65MA, the enzyme produced by recombinant E.
coli, was highly resistant to inactivation by a-haloke-
tones, except for the case of 4-bromo-3-oxobutano-

Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA
Table 3. Resistance of FDH to -Haloketones
2151
a
Remaining activity of FDH (
z
)
Compound
Thiobacillus sp. strain
Ancylobacter aquaticus strain
Candida boidinii
KNK65MA
KNK607M
ATCC32195
None
101.4
100.7
24.1
100.0
100.0
97.9
101.4
20.1
0
46.2
2.5
27.0
6.1
101.3
14.3
0
38.2
5.4
7.4
1.5
Ethyl 4-chloro-3-oxobutanoate
Ethyl 4-bromo-3-oxobutanoate
1-chloro-2-oxopropane
Ethyl 2-chloro-3-oxobutanoate
2, 3?-dichloroacetophenone
2-chloro-1-(3-pyridyl)-ethanone
90.1
The cell-free extracts prepared as described in Materials and Methods were mixed with an equal amount of 20 m
a-haloketones dissolved in 100 m potassi-
M M
um phosphate buŠer (pH 6.5), incubated for 5 h at 30 C, and then assayed. The resistance to -haloketones was expressed as a percentage of the remaining activ-
9
a
ity after the incubation compared to that before the incubation.
ate. Compared to the results with the enzyme, the
decreased in number. In addition, the mutant FDH
of Pseudomonas containing a Cys256Ser or a
Cys256Met replacement have been reported to have
increased resistance to inactivation by oxidation and
SH reagents.26) These ˆndings suggest the importance
of the 256th residue being Val, in the case of the FDH
of strain KNK65MA, in order to achieve this type of
resistance. On the other hand, Candida FDH25) con-
tains only two Cys residues, at the 23rd and 262nd
positions, which correspond to the 53rd and 289th
FDHs of Ancylobacter17) and Candida25) were inacti-
vated to a much greater extent by a-haloketones. The
higher stability of the strain KNK65MA FDH was
unique to this type of FDH; this feature proved
important for the regeneration of the cofactor in the
enzymatic conversion of
a-haloketones to chiral a-
halohydrins, which are important synthetic inter-
mediates for pharmaceuticals.
One of the reasons for the observed resistance to a-
haloketones may have been the replacement of the
Cys residues of the FDH, which is not the case with
positions of bacterial FDHs, respectively. Slusarczyk
25)
et al
.
have reported that the Cys23 (the 53rd posi-
the other FDHs, as shown in Fig. 3. Because
a
-
tion in bacterial FDH) of the unstable wild-type
Candida FDH was replaced by Ser by site-directed
mutagenesis; in that study, the stability in terms of
oxidative stress was increased. Based on this report,
the 53rd residue of bacterial FDHs being Ser, as is the
case in the FDH of strain KNK65MA, is also im-
portant for stability. In addition to the potential
eŠects of the 53rd and the 256th residues, there may
be other explanations for the observed resistance to
haloketones are considered to react with nucleophilic
functional groups such as the SH group,41,42) and
therefore a-haloketones may react with the free SH
groups of the enzyme and may thereby inactivate it.
Existence of the free SH groups in Pseudomonas
FDH has been suggested previously.8) Replacement
of Cys residues may in this manner reduce the reac-
tivity between the enzyme and a-haloketones. Other
bacterial FDHs17–24) contain seven Cys residues at the
6th, 146th, 183rd, 249th, 256th, 289th, and 355th
positions; however, in the case of strain KNK65MA,
Cys residues at the 256th and the 289th positions are
replaced by Val and Ala, respectively. Furthermore,
a-haloketones, such as an eŠect of the 289th residue,
or the in‰uence of other non-identical amino acid
residues.
In conclusion, we obtained
FDH, which was found to be suitable for the eco-
nomical production of chiral -halohydrins, when
used as an enzyme for cofactor regeneration.
a-haloketone-resistant
the FDH of strain KNK65MA was more resistant to
a
17)
SH reagents than that of Ancylobacter
,
suggesting
that the reactive SH groups on the enzyme were

2152
H. NANBA et al.
Puriˆcation and characterization of formate de-
hydrogenase in a methanol-utilizing yeast, Kloeckera
sp. No. 2201. Agric. Biol. Chem., 38, 111–116 (1974).
Acknowledgments
13) Schutte, H., Flossdorf, J., Sahm, H., and Kula, M.-
R., Puriˆcation and properties of formaldehyde
dehydrogenase and formate dehydrogenase from
We would like to thank Dr. S. Takahashi of the
Fine Chemicals Division, Kaneka Corporation, for
his advice and support during the course of this
study. We also wish to thank Ms. H. Nakanishi of
the Fine Chemicals Research Laboratories, Kaneka
Corporation, for her assistance with this study.
Candida boidinii. Eur. J. Biochem., 62, 151–160
(1976).
14) Van Dijken, J. P., Oostra-Demkes, G. J., Otto, R.,
and Harder, W., S-formylglutathione: the substrate
for formate dehydrogenase in methanol-utilizing
yeast. Arch. Microbiol., 111, 77–83 (1976).
References
15) Izumi, Y., Kanzaki, H., Morita, S., Futazuka, H.,
and Yamada, H., Characterization of crystalline
1) Crosby, J., Chirality in industry-an overview. In
``Chirality in Industry'', eds. Collins, A. N.,
Sheldrake, G. N., and Crosby, J., John Wiley &
Sons, Chichester, pp. 1–66 (1992).
formate dehydrogenase from Candida methanolica
Eur. J. Biochem., 182, 333–341 (1989).
.
16) Avilova, T. V., Egorova, O. A., Ioanesyan, L. S.,
and Egorov, A. M., Biosynthesis, isolation and prop-
erties of NAD-dependent formate dehydrogenase
2) Beck, G., Jendralla, H., and Kesseler, K., Practical
large scale synthesis of tert-butyl (3
3,5-
R,5S)-6-hydroxy-
O
-isopropylidene-3,5-dihydroxyhexanoate: essen-
from the yeast Candida methylica
152, 657–662 (1985).
. Eur. J. Biochem.,
tial building block for HMG-CoA reductase inhibi-
tors. Synthesis, 1014–1018 (1995).
17) Nanba, H., Takaoka, Y., and Hasegawa, J.,
Puriˆcation and characterization of formate de-
hydrogenase from Ancylobacter aquaticus strain
KNK607M, and cloning of the gene. Biosci.
Biotechnol. Biochem., 67, 720–728 (2003).
3) Zhou, B.-N., Gopalan, A. S., VanMiddlesworth, F.,
Shieh, W.-R., and Sih, C. J., Stereochemical control
of yeast reductions. 1. Asymmetric synthesis of
L
-
Carnitine. J. Am. Chem. Soc., 105, 5925–5926
(1983).
18) Muller, U., Willnow, P., Rusching, U., and Hopner,
T., Formate dehydrogenase from Pseudomonas ox-
4) Yasohara, Y., Kizaki, N., Hasegawa, J., Wada, M.,
Kataoka, M., and Shimizu, S., Stereoselective reduc-
tion of alkyl 3-oxobutanoate by carbonyl reductase
alaticus. Eur. J. Biochem., 83, 485–498 (1978).
19) Egorov, A. M., Avilova, T. V., Dikov, M. M.,
Popov, V. O., Rodionov, Y. V., and Berezin, I. V.,
NAD-dependent formate dehydrogenase from
methylotrophic bacterium, strain 1. Eur. J.
Biochem., 99, 569–576 (1979).
from Candida magnoliae
12, 1713–1718 (2001).
. Tetrahedron: Asymmetry,
5) Wong, C.-H., and Whitesides, G. M., Enzyme-cata-
lyzed organic synthesis: NAD(P)H cofactor regenera-
tion by using glucose 6-phosphate and the glucose-6-
phosphate dehydrogenase from Leuconostoc mesen-
20) Asano, Y., Sekigawa, T., Inukai, H., and Nakazawa,
A., Puriˆcation and properties of formate de-
hydrogenase from Moraxella sp. strain C-1. J.
Bacteriol., 170, 3189–3193 (1988).
21) Iida, M., Kitamura-Kimura, K., Maeda, H., and
Mineki, S., Puriˆcation and characterization of a
NAD＋-dependent formate dehydrogenase produced
by Paracoccus sp. Biosci. Biotechnol. Biochem., 56,
1966–1970 (1992).
22) Tishkov, V. I., Galkin, A. G., Marchenko, G. N.,
Tsygankov, Y. D., and Egorov, A. M., Formate
dehydrogenase from methylotrophic bacterium Pseu-
domonas sp. 101: gene cloning and expression in
Escherichia coli. Biotechnol. Appl. Biochem., 18,
201–207 (1993).
23) Shinoda, T., Satoh, T., Mineki, S., Iida, M., and
Taguchi, H., Cloning, nucleotide sequencing, and ex-
pression in Escherichia coli of the gene for formate
dehydrogenase of Paracoccus sp. 12-A, a formate-as-
teroides. J. Am. Chem. Soc., 103, 4890–4899 (1981).
6) Wong, C.-H., Drueckhammer, D. G., and Sweers,
H. M., Enzymatic vs. fermentative synthesis: ther-
mostable glucose dehydrogenase catalyzed regenera-
tion of NAD(P)H for use in enzymatic synthesis. J.
Am. Chem. Soc., 107, 4028–4031 (1985).
7) Wong, C.-H., and Whiteside, G. M., Enzyme-cata-
lyzed organic synthesis: NAD(P)H cofactor regenera-
tion using ethanol alcohol dehydrogenase aldehyde
W
W
dehydrogenase and methanol alcohol dehydrogenase
W
aldehyde dehydrogenase formate dehydrogenase. J.
W
W
Org. Chem., 47, 2816–2818 (1982).
8) Popov, V. O., and Lamzin, V. S., NAD＋-dependent
formate dehydrogenase. Biochem. J., 301, 625–643
(1994).
9) Ferry, J. G., Formate dehydrogenase. FEMS
Microbiol. Rev., 87, 377–382 (1990).
10) Ohyama, T., and Yamazaki, I., Puriˆcation and
some properties of formate dehydrogenase. J.
Biochem., 75, 1257–1263 (1974).
11) Colas des Francs-Small, C., Ambard-Bretteville,
F., Small, I. D., and Remy, R., Identiˆcation of a
major soluble protein in mitochondria from non-
photosynthetic tissues as NAD-dependent formate
similation bacterium. Biosci. Biotechnol. Biochem.
66, 271–276 (2002).
,
24) Galkin, A., Kulakova, L., Tishkov, V., Esaki, N.,
and Soda, K., Cloning of formate dehydrogenase
gene from a methanol-utilizing bacterium Mycobac-
terium vaccae N10. Appl. Microbiol. Biotechnol., 44,
479–483 (1995).
25) Slusarczyk, H., Felber, S., Kula, M.-R., and Pohl,
M., Stabilization of NAD-dependent formate de-
hydrogenase from Candida boidinii by site-directed
dehydrogenase. Plant Physiol.
, 102, 1171–1177
(1993).
12) Kato, N., Kano, M., Tani, Y., and Ogata, K.,

Formate Dehydrogenase from Thiobacillus sp. Strain KNK65MA
2153
mutagenesis of cysteine residues. Eur. J. Biochem.
,
motolerant
N
-carbamyl-
D
-amino acid amidohydro-
267, 1280–1289 (2000).
lase by recombinant Escherichia coli
Bioeng., 87, 149–154 (1999).
34) Sanger, F., Nicklen, S., and Coulson, A. R., DNA
sequencing with chain-terminating inhibitiors. Proc.
Natl. Acad. Sci. USA, 74, 5463–5467 (1977).
. J. Biosci.
26) Tishkov, V. I., Galkin, A. G., Marchenko, G. N.,
Egorova, O. A., Sheluho, D. V., Kulakova, L. B.,
Dementieva, L. A., and Egorov, A. M., Catalytic
properties and stability of a Pseudomonas sp. 101
formate dehydrogenase mutants containing Cys-255-
Ser and Cys-255-Met replacements. Biochem.
Biophys. Res. Commun., 192, 976–981 (1993).
27) Rojkova, A. M., Galkin, A. G., Kulakova, L. B.,
Serov, A. E., Savitsky, P. A., Fedorchuk, V. V., and
Tishkov, V. I., Bacterial formate dehydrogenase. In-
creasing the enzyme thermal stability by hydrophobi-
zation of alfa-helices. FEBS Lett., 445, 183–188
(1999).
28) Sambrook, J., Fritsch, E. F., and Maniatis, T.,
Molecular cloning, A laboratory manual, 2nd ed.,
Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York (1989).
29) Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and
Randall, R. J., Protein measurement with the Folin
phenol reagent. J. Biol. Chem., 193, 265–275 (1951).
30) Laemmli, U. K., Cleavage of structural proteins
during the assembly of bacteriophage T4. Nature
227, 680–685 (1970).
31) Triglia, T., Peterson, M. G., and Kemp, D. J., A
procedure for in vitro ampliˆcation of DNA segments
that lie outside the boundaries of known sequences.
Nucleic Acids Res., 16, 8186 (1988).
32) ``Current Protocols in Molecular Biology'', eds.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore,
D. D., Seidman, J. G., Smith, J. A., and Struhl, K.,
Current Protocols, unit 2.4 (1987).
33) Nanba, H., Ikenaka, Y., Yamada, Y., Yajima, K.,
Takano, M., and Takahashi, S., Production of ther-
35) Taylor, B. F., and Hoare, D. S., New facultative
Thiobacillus and a reevaluation of the heterotrophic
potential of Thiobacillus novellas. J. Bacteriol., 100,
487–497 (1969).
36) Chandra, T. S., and Shethna, Y. I., Oxalate,
formate, formamide, and methanol metabolism in
Thiobacillus novellas. J. Bacteriol., 131, 389–398
(1977).
37) Wood, A. P., and Kelly, D. P., Mixotrophic growth
of Thiobacillus A2 in chemostat culture on formate
and glucose. J. Gen. Microbiol., 125, 55–62 (1981).
38) Smith, N. A., and Kelly, D. P., Mechanism of oxida-
tion of dimethyl disulphide by Thiobacillus thioparus
strain E6. J. Gen. Microbiol., 134, 3031–3039 (1988).
39) Pronk, J. T., de Bruijin, P., van Dijken, J. P., Bos,
P., and Kuenen, J. G., Energetics of mixotrophic and
autotrophic C1-metabolism by Thiobacillus acidophi-
,
lus. Arch. Microbiol., 154, 576–583 (1990).
40) Saleeba, J. A., Cobbett, C. S., and Hynes, M. J.,
Characterization of the amdA-regulated aciA gene of
Aspergillus nidulans. Mol. Gen. Genet., 235, 349–358
(1992).
41) Teich, S., and Curtin, D. Y., The alkaline cleavage of
desyl thioethers. J. Am. Chem. Soc., 72, 2481–2483
(1950).
42) Terasawa, T., and Okada, T., Novel heterocyclic syn-
thons, synthesis and properties of thia- and oxacyclo-
hexane-3, 5-diones. J. Org. Chem., 42, 1163–1169
(1977).