Investigation new positions for catalytic activity of Chaetomium thermophilum and Ceriporiopsis subvermispora formate dehydrogenases

Article abstract of DOI:10.1080/10242422.2020.1863951

NAD⁺-dependent formate dehydrogenases (FDHs, E.C 1.2.1.2) catalyse the reversible reaction of CO₂ to formate ion (HCOO^?) and reduces NAD⁺ molecule to NADH. Previously described FDHs from Chaetomium thermophilum (CtFDH) and Ceriporiopsis subvermispora (CsFDH) are active against formate and HCO₃^–. In this study, we examined the functional effects of active site mutations in Ct and Cs NAD⁺-dependent FDHs. The residues Ile94, Asn120, Val310, His312 at CtFDH and Asn312, Val313, Val331 at CsFDH are located in the active site. The effects of amino acid changes on catalytic properties and thermal stability of CtFDH and CsFDH revealed some interesting results compared with structurally equivalent positions that have been studied in the literature. The strongest effect was observed in CsFDH Val313Pro against HCO₃^–. The K_M value of the CsFDH Val313 enzyme for HCO₃^– substrate dramatically decreased, and enzyme activity increased. In CtFDH mutants, all enzymes except Val310Asn showed an increased k_cat value when K_M values increase. Analyses of results of mutant CtFDHs and CsFDHs give some promising results for CO₂ reduction as compared to the literature. Structural analyses of the substrate-binding site were done by homology modelling.

Full text of DOI:10.1080/10242422.2020.1863951

Biocatalysis and Biotransformation
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Investigation new positions for catalytic activity
of Chaetomium thermophilum and Ceriporiopsis
subvermispora formate dehydrogenases
Kübra Koçdemir, Fatma Şen, Yasin Adem Wedajo, Muazzez Çağla Bilgici,
Mustafa Bayram, İlke Selçuk, Berin Yılmazer, Mehmet Mervan Çakar, Elif
Sibel Aslan & Barış Binay
To cite this article: Kübra Koçdemir, Fatma Şen, Yasin Adem Wedajo, Muazzez Çağla Bilgici,
Mustafa Bayram, İlke Selçuk, Berin Yılmazer, Mehmet Mervan Çakar, Elif Sibel Aslan & Barış
Binay (2020): Investigation new positions for catalytic activity of Chaetomiumꢀthermophilum and
Ceriporiopsisꢀsubvermispora formate dehydrogenases, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2020.1863951
To link to this article: https://doi.org/10.1080/10242422.2020.1863951
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2020.1863951
RESEARCH ARTICLE
Investigation new positions for catalytic activity of Chaetomium
thermophilum and Ceriporiopsis subvermispora formate dehydrogenases
a
a
b
c
c
ꢀ
ꢀ
€
ꢀ
Kubra Koc¸demir , Fatma S¸en , Yasin Adem Wedajo , Muazzez C¸agla Bilgici , Mustafa Bayram ,
a
a
a
d
e
_
Ilke Selc¸uk , Berin Yılmazer , Mehmet Mervan C¸akar , Elif Sibel Aslan and Barıs¸ Binay
b
aDepartment of Molecular Biology and Genetics, Gebze Technical University, Gebze, Kocaeli, Turkey; Department of Chemistry,
c
Gebze Technical University, Gebze, Kocaeli, Turkey; Department of Biotechnology, Gebze Technical University, Gebze, Kocaeli,
d
e
_
Turkey; Department of Molecular Biology and Genetics, Biruni University, Topkapı, Istanbul, Turkey; Department of Bioengineering,
Gebze Technical University, Gebze, Kocaeli, Turkey
ABSTRACT
ARTICLE HISTORY
NADþ-dependent formate dehydrogenases (FDHs, E.C 1.2.1.2) catalyse the reversible reaction of
CO2 to formate ion (HCOOꢁ) and reduces NADþ molecule to NADH. Previously described FDHs
from Chaetomium thermophilum (CtFDH) and Ceriporiopsis subvermispora (CsFDH) are active
Received 3 November 2020
Revised 8 December 2020
Accepted 9 December 2020
–
against formate and HCO3 . In this study, we examined the functional effects of active site
KEYWORDS
mutations in Ct and Cs NADþ-dependent FDHs. The residues Ile94, Asn120, Val310, His312 at
CtFDH and Asn312, Val313, Val331 at CsFDH are located in the active site. The effects of amino
acid changes on catalytic properties and thermal stability of CtFDH and CsFDH revealed some
interesting results compared with structurally equivalent positions that have been studied in the
Protein engineering; NADþ-
dependent formate
dehydrogenase; CO2
reduction; site-directed
mutagenesis; molecu-
lar modelling
–
literature. The strongest effect was observed in CsFDH Val313Pro against HCO3 . The KM value of
the CsFDH Val313 enzyme for HCO3 substrate dramatically decreased, and enzyme activity
–
increased. In CtFDH mutants, all enzymes except Val310Asn showed an increased kcat value
when KM values increase. Analyses of results of mutant CtFDHs and CsFDHs give some promis-
ing results for CO2 reduction as compared to the literature. Structural analyses of the substrate-
binding site were done by homology modelling.
Introduction
Since the formed CO2 can be easily removed from the
reaction environment, NADH is obtained purely. Until
now, FDHs have been evaluated only as excellent
NADH regenerators, although recent studies have
shown that some FDHs have the ability to reduce car-
bon dioxide to formate as a reverse reaction (Lu et al.
2006, Alissandratos et al. 2013, Aslan et al. 2017, Pala
et al. 2018).
Increased atmospheric CO2-concentration is widely
being considered the main driving factor of global
warming (Florides and Christodoulides 2009). Increases
in human population, industrial activity, and natural
disasters (volcanic eruptions, etc.) are the major causes
of CO2 emission (C¸akar et al. 2020). Climate change is
a global challenge that has no borders; coordinated
work by all countries is required to combat it (Glueck
et al. 2010). It has been predicted that by 2050 the
CO2 concentration in the atmosphere will reach over
500 ppm, nearly double the concentration present
NADþ-dependent formate dehydrogenases (FDHs) (E.C
1.2.1.2) catalyse formate oxidation to carbon dioxide
(CO2) by the reduction of NADþ to NADH (Tishkov
and Popov 2004). In a formate oxidation reaction, a
single carbon–hydrogen bond is broken in the formate
and a new carbon–hydrogen bond is formed in the
NADH, producing CO2 (Schiøtt et al. 1998). NADþ-
dependent FDHs are mostly seen in industrial regener-
ation systems, where they are usually used for the
synthesis of valuable optically active chiral compounds
such as drugs and amino acids (Hatrongjit and
Packdibamrung 2010). Among the enzymes used for
cofactor regeneration, formate dehydrogenase has
been reported as the most suitable due to having
many advantages in the recycling process of NADH:
an appropriate thermodynamic equilibrium, the immo-
bility of the substrate, and an inert by-product (CO2).
CONTACT Barıs¸ Binay
These authors contributed equally.
This article has been republished with minor changes. These changes do not impact the academic content of the article.
binay@gtu.edu.tr
ꢀ
Supplemental data for this article can be accessed here.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
K. KOÇDEMIR ET AL.
before the industrial revolution (280 ppm) (Yuan et al.
2019). Biotransformation of CO2 has the benefit of glo-
bal warming reduction and also is a powerful alterna-
tive to produce renewable energy (C¸akar et al. 2020).
Because of the ability of biocatalysts to efficiently cata-
lyse processes under mild conditions with limited by-
product formation, they play a critical role in the
transformation of CO2 by lowering activation barriers
(Alissandratos et al. 2013, Choe et al. 2014). However,
only a few biocatalysts, such as formate dehydrogen-
ase, can capture CO2. The reverse reaction of FDHs
has promising potential for methods of carbon fixation
from the atmosphere and the oceans. Formate, which
can be produced from CO2 as a feedstock, is also an
economically and environmentally valuable chemical
(Alissandratos et al. 2013, 2014, Hawkins et al. 2013).
Our previous results showed that NADþ/NADH-
dependent FDHs show low catalytic efficiency for the
reduction of CO2 (Aslan et al. 2017). Also, regeneration
of cofactors requires that FDHs have stability in
extreme process conditions and high cofactor turnover
rates. However, the reported FDHs have limited prop-
erties such as thermostability and catalytic efficiency
in order to fulfil the industrial requirements (Labrou
and Rigden 2001, Galkin et al. 2002, Leopoldini et al.
2008, Alissandratos et al. 2013, Wu et al. 2013,
Bassegoda et al. 2014, Choe et al. 2014, Aslan et al.
2017, Maia et al. 2017). These enzymes would need to
be engineered before they could be used at an indus-
trial scale for the conversion of CO2 to hydrocarbons
and to have more efficient cofactor regeneration. For
protein engineering improvements, study of the struc-
ture–function relationships at the FDH active site, and
especially the catalytic mechanism, is essential.
substitution makes this position worth to investigate.
Also, previously identified three significant residues
(C¸akar et al. 2020, Yilmazer et al. 2020) were selected
for the alanine scanning mutagenesis in order to sup-
port previous studies. For this purpose, desired muta-
tions were introduced into the active site of the FDHs
from Cs and Ct. The kinetic parameters were deter-
mined, and the results were investigated with hom-
ology modelling and enzyme–substrate/cofactor
docking studies to provide a further understanding of
the forward and reverse catalytic mechanism of
the FDHs.
Materials and methods
General equipment and chemicals
All chemicals were purchased from Sigma-Aldrich.
DH5a competent cells, BL21 competent cells, and the
pET 45 b (þ) plasmid were purchased from Sigma
Aldrich and the 6xHis-tag protein purification column
was a BabyBio purchased from BioWorks. The site-
directed mutagenesis was carried out under PCR with
a Techne Prime Thermal Cycler purchased from
Techne. The plasmid isolation kit was purchased from
NucleoSpin Plasmid (NoLid), and a mini kit for plasmid
DNA purification was purchased from Macherey-Nagel.
The centrifuge was Beckman Coulter purchased from
Sigma, and the benchtop centrifuge was purchased
from Allegra. The gel-screening blue light was from
Serva, and the absorbance microplate reader and
microplate spectrometer Epoch2 was purchased
from BioTek.
Bioinformatic analysis
Although FDH enzymes have conserved amino acid
positions that form as catalytic sites, they reveal differ-
ent kinetic rates for the same substrate (Tishkov and
Popov 2004). Single-amino acid changes in the active
sites of enzymes can significantly alter the stability,
catalytic efficiency, and substrate specificity of the
The CsFDH was aligned with well-known FDHs and
three non-aligned residues were selected for mutation
based on their relative proximity to the active site
(Figure 1). The three candidates, Asn312, Val313, and
Val331 residues in 2NAD numbering that close to the
active site, were identified. The crystal structures of
CtFDH (PDB:6T8Y) and previous studies were used as a
guide to the selection of CtFDH. Previously identified
Ile94, Asn120 and His312 residues were selected for
Alanine substitution. The oligonucleotide primers were
to confer the desired mutations in the CtFDH and
CsFDH plasmids.
€
enzyme (Karimaki et al. 2004, Kim et al. 2014, Lenz
et al. 2018). Therefore, we investigated how specific
mutations could affect the kinetic properties of FDH
enzyme. In order to examine the catalytic site of
CsFDH, it was aligned with well-known FDHs and three
non-aligned positions were selected for mutation
based on their relative proximity to the active site.
CtFDH also has one of these three amino acid substi-
tutions which is Thr to Val substitution on the same
position. Although CsFDH and CtFDH have shown
quite different catalytic characteristics as stated in pre-
vious studies (Aslan et al. 2017), the common
Improving the purification of CsFDH and CtFDH
The desired mutations were incorporated by an overlap-
ping polymerase chain reaction (PCR) method using

BIOCATALYSIS AND BIOTRANSFORMATION
3
Figure 1. Alignment of formate dehydrogenases. Symbols: Ps, Pseudomonas sp. (strain 101) (PseFDH); Cb, Candida boidinii
(CbFDH); Ts, Thiobacillus sp. (strain KNK65MA) (TsFDH); Cm, Candida methylica (CmFDH); Ct, Chaetomium thermophilum (CtFDH).
Table 1. List of primers used for site-directed mutagenesis.
Source of FDH
Name
Sequence (50 to 30)
Ct
Ile94Ala _fw
ACTGGCGGTGACCGCCGGTGCCGGCAGCGATCAT
GCTGGTTAATACGGCGGCCGGCGCTATCGTCGTG
GTGGCAATGCTATGAACCCGCACATGAGCG
GGCAATGCTATGGTACCGGCCATGAGCGGCACCA
GGTGGCAATGCTATGACCCCGCACATGAGCG
GCAGGCGATGTGTGGTTTGTTCAGCCGGCG
GGCGATGTGTGGAATCCGCAGCCGGCGC
Asn120Ala_fw
Val310Asn_fw
His312Ala_fw
Val310Thr_fw
Asn312Phe_fw
Val313Pro_fw
Val331Thr_fw
Cs
GTGGCAATGGTATGACCCCGCACTATAGCG
complementary oligonucleotides. Primers ordered from
Eurofins and their sequences are listed in Table 1. The
wild-type CtFDH and CsFDH genes were subjected to
site-directed mutagenesis according to the standard PCR
method (98 ꢂC for 30 seconds, 98 ꢂC for 10 seconds,
65 ꢂC for 20 seconds, 72 for 2.5 minutes, 72 ꢂC for
7 minutes for the final extension for one cycle) in the
presence of 1 mL dNTPs mix, 2,5mL primer mix, 0,5mL
Phusion High Fidelity DNA polymerase in a total 50 mL
reaction. The amplified PCR product involves 6xHis-tag
at the C-terminal and N-terminal of the pET45b (þ)
expression plasmid. After PCR, 1 lL of the DpnI restric-
tion enzyme was added into the PCR product and incu-
bated at 37 ꢂC for 2hours. DpnI-treated PCR products
were confirmed on 1% agarose gel electrophoresis. The
mutated product was transformed into E. coli DH5a
competent cells for gene cloning and selected on Luira-
Broth (LB) agar containing 100 mg/ml ampicillin. The final
constructed product was sequenced in Eurofins MWG
Operon. All of the mutation positions progressed under
the same cloning and mutation procedure as described.
coli BL21 protein expression vectors, were grown over-
night (16–18 h) on a rotary shaker in LB (ꢃ10–15 mL)
supplemented with ampicillin (100 mg/mL). Previously
prepared Studier Media (autoinduction) involved 4 mL
LB media containing the culture and 100 mg/mL ampi-
cillin incubated overnight (16 h) on a rotary shaker
(Studier 2005). After that, the cells were harvested by
centrifugation (15,000 ꢄ g, 20 min, þ4 ꢂC), and the
resulting cell pellet was stored at ꢁ80 ꢂC.
The wild type and mutated proteins were purified
by His-tag affinity column using nickel nitrilotriacetic
acid (Ni-NTA) resin (supplied with the kit). The pelleted
cells were kept at 37 ꢂC for 30 min and then resus-
pended in 10 mL of buffer A containing 20 mM
NaH2PO4, 500 mM NaCl and 30 mM imidazole, with the
pH adjusted to 7.4. Lysozyme (1 mg/mL) was added to
the cell suspension, PMSF (phenylmethylsulphonyl
fluoride, 4 mL/g) was added to the cell suspension and
the resulting mixture was incubated on ice for 30 min.
Each incubated pellet was sonicated to break up the
cells to obtain extracellular extract for further purifica-
tion studies. A sample (50 mL) was collected at each
step to determine the total protein content. The
remaining cell suspension was centrifuged at
15,000 ꢄ g for 15 min at 4 ꢂC.
Expression and purification of Wild-Type and
mutant recombinant CtFDH and CsFDH proteins
The obtained and confirmed mutant and wild type
CtFDH and CsFDH genes were expressed in E. coli
BL21 (DE3) cells for the production and purification of
the corresponding proteins. Bacterial cultures involv-
ing the desired plasmid, which was transformed in E.
The 6xHis-tag proteins were purified on Ni-NTA
HisTrap column chromatography using a linear gradi-
ent of 50–250 mM imidazole in a buffer consisting of
200 mM sodium phosphate buffer (NaPi), 500 mM
NaCl pH 7.4. The fractions were analysed with sodium

4
K. KOÇDEMIR ET AL.
dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE). Purified proteins were concentrated with
30 kDa cut-off ultracentrifuge filter tubes. The purified
proteins were quantitated with the bicinchoninic acid
assay (Smith et al. 1985).
CtFDH (PDB ID: 6T8Y) (Yilmazer et al. 2020). Formate
and hydrogen carbonate substrates were docked into
CsFDH and CtFDH (hydrogen carbonate only) active
site pockets in silico via the AutoDock Vina (Trott and
Olson 2010). Substrate binding pocket drawings were
done by PyMOL (Molecular Graphics System, Version
2.0 Schrodinger, LLC), 2D cofactor binding domain
drawings were done by PoseView (Stierand and Rarey
2010), and rotamers of mutated positions were pre-
dicted with UCSF Chimaera (Pettersen et al. 2004,
Shapovalov and Dunbrack Jr. 2011).
pH assay
The relative activity of wild type and all the mutant
FDHs were measured in different 100 mM buffers for
different pH ranges at 25 ꢂC in a solution containing
10 mM formic acid buffer and 1.0 mM NADþ. The
selected 100 mM buffers were citrate for pH 3.0–6.0,
NaPi for pH 6.0–7.5, Tris–HCl for pH 7.5–9.0, Glycine
for pH 9.0–10.0, sodium carbonate pH 9.5–11.0 and
KCl-NaOH pH 11.0–13.0.
Results and discussion
Expression and purification
Site-directed mutagenesis was used to construct muta-
tions at selected positions in the CtFDH and CsFDH
genes. The desired mutations were confirmed by DNA
sequencing. FDH protein variants containing the N-ter-
minal 6xHis-tag were isolated by Ni-NTA HisTrap col-
umn chromatography. The fractions were analysed
with SDS-PAGE (Supplementary Figure S1) and pure
fractions were collected. Wild-type and the mutant
enzymes were obtained with high yield.
Thermostability assay
The thermostability of enzymes was measured by
incubating the purified FDHs for 15 min at tempera-
tures from 25 ꢂC to 65 ꢂC. The residual activity of the
incubated samples was measured in specific activity
assays at 24 ꢂC. The specific activity of FDHs was meas-
ured in optimum 100 mM buffers containing 20 mM
sodium formate and 1.0 mM NADþ for formate oxida-
tion or 20 mM sodium bicarbonate and 0.5 mM NADH
for CO2 reduction.
pH and thermostability
The relative thermostability of the enzymes was calcu-
lated by the ratio of initial rate constants of wild-type
and the mutants (Tables 2 and 3). The initial rate con-
stant derived from inactivation rate constant which is
used to determine thermal stability. The dependency
of the inactivation rate constant is determined by
transition state theory (Tishkov and Popov 2006). The
relative thermal stability is determined by the ratio of
initial rate constants for the initial enzyme and its
Enzyme kinetic assays
Kinetic parameters of wild type and all the mutant
FDHs were determined through monitoring the
change in absorbance at 340 nm due to NADH forma-
tion or consumption in 100 mM of optimum buffers
for each FDH. The reaction mixtures contained sodium
formate and 1.0 mM NADþ for formate oxidation and
sodium bicarbonate and 0.5 mM NADH for CO2 reduc-
tion. The substrate concentration ranges for the differ-
ent assays were 0.5–50 mM for formate, 0.5–40 mM for
sodium bicarbonate. All reactions were performed at
25 ꢂC. The Michaelis–Menten kinetic constants were
determined from triplicate measurements of the initial
velocities using GraphPad Prism software.
Table 2. Relative thermostability of CtFDH mutants against
formate and hydrogen carbonatea.
Substrates
Formate
Ct
Hydrogen carbonate
Temperature
(ꢂC)
Ct
Ct
Ct
Ct
Ct
I94A V310N V310T H312A
I94A
V310N
25
30
35
40
45
50
55
60
65
2.6
1.0
0.5
2.0
2.9
4.8
3.3
0
4.8
5.4
1.9
1.5
1.7
1.3
3.2
0
4.6
5.8
4.6
4.0
4.6
2.7
0.3
2.9
2.9
2.5
2.2
2.1
1.6
1.2
1.3
1.2
0
6.5
5.2
0.9
0.5
2.1
5.7
5.9
0
7.3
8.8
7.9
1.8
6.6
6.7
6.7
0
Molecular modelling
The 3D homology model of CsFDH was generated in
Swiss-Model (Arnold et al. 2006) using the published
structure of Pseudomonas sp. 101 formate dehydrogen-
ase (PseFDH, PDB ID: 2NAD) (Lamzin et al. 1994) as a
template, and the published structure was used for
0
0
0
0
0
aThe initial rate constants that used for thermal stability calculation are
wild type enzyme measured at 55 ꢂC against formate and at 40 ꢂC for
CO2 reduction.

BIOCATALYSIS AND BIOTRANSFORMATION
5
mutant (kinini/kinmut). Due to their direct relation to the
activities, the lower values indicate more sta-
ble enzymes.
The optimum pH rates of CsFDH, CtFDH, and their
mutants for formate oxidation and CO2 reduction are
shown in Table 4. The measurements show that the
optimum pH for formate oxidation is 6.5 for the wild-
type, Asn312Phe and Val313Pro mutants, whereas 6.0
is the pH optimum for the Val331Thr mutant of
CsFDH. The Val331Thr mutant showed higher activity
towards formate than the wild-type CsFDH between
pH 6.0–10.0. Although they show similar patterns
between pH 6.0 and 10.0, the formate oxidation activ-
ity of the mutant is higher than the wild type in acidic
pH (4.5, 5.0). On the other hand, no significant differ-
ences in pH optimum were found for the CsFDH-
Val313Pro and CsFDH-Asn312Phe mutants. The opti-
mum pH for formate oxidation is 7.0 for the wild-type
CtFDH and His312Ala, whereas the optimum pH is 7.5,
5.5, and 5.0 for the Ile94Ala, Val310Asn and Val310Thr
mutants, respectively. While wild-type CtFDH and
His312Ala show similar patterns, the mutant has a
slightly higher activity for formate oxidation. CtFDH-
Val310Asn shows higher activity than wild type CtFDH
in acidic pH (5.0–7.0), while formate oxidation in
CtFDH-Ile94Ala is slightly higher in neutral pH.
Rate constants of wild-type enzymes were meas-
ured at 55 ꢂC in forward reaction (formate oxidation)
–
and 40 ꢂC against HCO3 for the CtFDH wild-type,
while it was measured at 45 ꢂC in forward reaction
–
and 30 ꢂC against HCO3 for the CsFDH wild-type. In
the formate-direction reaction, the CtFDH-Val310Thr
mutant is more stabilized than the wild-type at 55 ꢂC
and CtFDH-Ile94Ala mutant is more stabilized than
wild-type at 35 ꢂC, whereas other mutants did not
show any significant difference. At 35 ꢂC, CsFDH-
Asn312Phe mutants were more stable in formate oxi-
dation than the wild-type, but drastically lost stability
in CO2 reduction. The stability of the CtFDH-Ile94Ala
mutant was increased at 35 ꢂC and 40 ꢂC against
–
HCO3 , while CtFDH-Val310Asn mutants showed lower
stability than the wild-type. The Val313Pro mutant was
also more stabilized than its wild-type at 35 ꢂC
ꢁ
against HCO3
.
The measurements show that the optimum pH of
activity towards HCO3 is 6.0 for the wild-type CsFDH,
Table 3. Relative thermostability of CsFDH mutants against
–
formate and hydrogen carbonatea.
7.0 for Asn312Phe, and 6.5 for Val313Pro mutants.
Even though the wild-type and mutants show similar
patterns, the Asn312Phe mutant has lower CO2 reduc-
tion activity than the wild-type. The optimum pH
Substrates
Formate
Hydrogen carbonate
Cs
Cs
Cs
Cs
Cs
V313P
Temperature (ꢂC) N312F V313P V331T
N312F
–
25
30
35
40
45
50
55
60
65
1.8
1.0
0.9
2.7
28.5
211
0
6.2
6.1
5.8
5.3
4.6
4.1
24.8
46.9
0
1.5
1.5
1.3
1.1
1.2
3.8
19.2
97.4
0
10.3
2.4
4.6
5.5
11.9
10.7
30.8
0
2.2
1.0
0.8
1.7
1.9
2.1
5.1
0
towards HCO3 is 8.0 for the wild-type CtFDH, whereas
for the Ile94Ala and Val310Asn mutants, the optimum
pH is 6.0. Both CtFDH mutants show slightly higher
activity than wild-type in acidic pH (5.0–6.0).
0
0
0
0
Enzyme kinetic assays
aThe initial rate constants that used for thermal stability calculation are
wild type enzyme measured at 45 ꢂC for formate oxidation and at 30 ꢂC
for CO2 reduction.
Site-directed mutagenesis at non-conserved amino
acid positions in the active site was used to explore
Table 4. The kinetic constants of CtFDH and CsFDH wild type and mutant for hydrogen carbonate and formate.
Substrate
Formate
KM (mM)
3.22 0.460
Hydrogen carbonate
CtFDH
Optimum pH
kcat (sꢁ1
1.5 0.001
0.233 0.008 20.85 1.49
)
kcat/KM (sꢁ1mMꢁ1
)
Optimum pH
kcat (sꢁ1
)
KM (mM)
kcat/KM (sꢁ1mMꢁ1
)
Wild-Type
Ile94Ala
Asn120Ala
Val310Asn
His312Ala
Val310Thr
CsFDH
7.0
7.5
0.465 0.002
0.011 0.00
8
6
7.5
6
0.023 0.000
0.191 0.1
0.173 0.01
0.091 0.005
0.32 0.070
1.734 0.9
0.95 0.02
0.0690
0.110 0.11
0.164 0.01
0.072 0.012
a
5.5
7
5
0.136 0.009
0.100 0.000
0.387 0.028
1.58 0.69
7.68 0.56
6.80 1.90
0.086 0.013
0.013 0.000
0.057 0.014
1.25 0.403
a
a
Wild-Type
Asn312Phe
Val313Pro
Val331Thr
6.5
6.5
6.5
6
0.554 0.008
5.20 0.630
0.107 0.007
0.062 0.015
0.094 0.027
0.079 0.017
6
7
6.5
0.011 0.001 14.46 6.97
0.001 0.000
0.001 0.000
0.394 0.05
0.254 0.007 4.126 0.453
0.736 0.059 7.794 2.18
0.292 0.015 3.776 0.881
0.005 0.001
0.201 0.008
3.64 2.87
0.51 0.16
a
aNo activity

6
K. KOÇDEMIR ET AL.
the functional properties of these three positions in
CsFDH and CtFDH. The determined kinetic constants
are shown in Table 4 for wild-type CsFDH, CtFDH, and
their mutants.
efficiency in CO2 reduction than the semi-rationally
designed CtFDH mutant. The most promising mutant
studied for CO2 reduction, CtFDH-Gly93His/Ile94Ala,
ꢁ1
showed a slightly lower kcat/KM ratio (0.360 sꢁ1mM
)
The CsFDH Val331Thr mutation had higher KM and
kcat values, but low substrate affinity prevented an
increase in catalytic efficiency (kcat/KM decreased from
0.107 to 0.079 s/mM). The same mutation on CtFDH,
which is Val310Thr, had higher KM and lower kcat val-
ues, this mutation caused slightly decreasing in cata-
lytic efficiency. Though these two mutations did not
show activity at all in the CO2 reduction reaction and
CsFDH-Val331Thr had the lowest catalytic efficiency in
oxidizing formate, but the maximum speed of its cata-
lysed reaction can be reached with smaller amounts
of formate than the others. A different mutation on
the same position which is Val310Asn CtFDH mutation
resulted in decreased kcat and KM values but was fol-
lowed by a low catalytic efficiency due to a drastic
decrease in the kcat value.
than the CsFDH-Val313Pro mutant (C¸akar et al. 2020).
In addition, the site-directed mutations in CtFDH
were made separately in four positions in the active
site facing the substrate to study the possibility of
modifying the reaction by changing the enzyme–sub-
strate interactions. All three residues substituted with
Ala caused a decrease in catalytic efficiency. The
Ile94Ala and His312Ala CtFDH mutants had dramatic-
ally increased KM and decreased kcat for the forward
reaction. The mutations Ile94Ala and His312Ala
showed weakened binding of the substrate (KM values
of 20.85 mM and 7.18 mM, respectively). The
Asn120Ala mutation also showed a loss of activity in
formate oxidation. In addition to this dramatic effect,
the reverse reaction still continues. The 2.7-fold higher
KM than its wild-type indicates that the mutant had
decreased bicarbonate binding efficiency, while the
catalysis rate was increased from 0.25 to 1.73/s at the
same time. The mutation Ile94Ala showed the same
effect for the reverse reaction: the KM value increased
from 0.35 to 1.734, while the kcat value increased from
0.025 to 0.191. In contrast, the Ala mutation intro-
duced to His312, another substrate-binding site,
Although the CsFDH-Asn312Phe mutation slightly
increased the substrate affinity for formate, the cata-
lytic efficiency (kcat/KM) of this mutant decreased to
some degree, based on lowered kcat values (Table 4).
The CsFDH-Val313Pro mutant showed decreased
substrate affinity to formate (KM 5.2 to 7.8 mM),
whereas kcat was slightly increased. It had comparable
efficiency to the wild type in formate oxidation, while
CO2 reduction was four times as efficient as the wild
type and two times as efficient as CsFDH-Asn312Phe.
It also had more affinity towards hydrogen carbonate
than the two other enzymes. Even though CsFDH had
a very low activity for CO2 reduction (0.8 mU/mg), it
showed a dramatic increase over CtFDH in catalytic
caused
a
complete loss of activity in the
reverse reaction.
Molecular modelling of CsFDH and CtFDH
Despite CsFDH and CtFDH have ꢃ53% sequence iden-
tities with PseFDH, the first shell active site residues of
the three enzymes are conserved. Figures 2 and 3
highlight the substrate-binding pockets of CsFDH and
CtFDH which are built by residues Pro97, Phe98,
–
efficiency against HCO3 , which was found to be the
more promising FDH enzyme with its high activity for
CO2 reduction (34.6 mU/mg) (Choe et al. 2014, Aslan
et al. 2017). It even resulted in better catalytic
Figure 2. CsFDH active site residues are involved in the substrate-binding for formate oxidation and CO2 reduction reactions.

BIOCATALYSIS AND BIOTRANSFORMATION
7
Thr120, Gly124, Asn146, Val150, Arg284, Val309,
His332, and Gly335 (according to PseFDH numberings).
Formate and hydrogen carbonate substrates were
bound in the active site of CsFDH with H-bonds with
Val93, Asn117, Arg256, His309 and Gly312 (Val93-O
2.5 Å, Asn117-1HD21 2.1 Å, Arg256-HH11 2.0 and HH21
3.1 Å, His309-HE1 3.0 and HE2 2.4 Å, Gly312-HA3 3.9 Å
for formate and Val93-O 2.1 Å, Asn117-1HD2 2.1 Å,
Arg256-2HH2 2.6 Å, His309-HE2 2.5 Å and Gly312-CA
3.5 Å for hydrogen carbonate, Figure 2).
The mutations Phe311Asn in PseFDH and
Phe290Asn in SoyFDH similarly showed a decrease in
catalytic efficiency due to their loss of affinity against
formate (Alekseeva et al. 2012, Tishkov et al. 2015).
The mutations studied on PseFDH-Phe311 by Tishkov
et al. revealed the formation of additional H-bonds.
PseFDH wild-type forms only one H-bond in between
Phe311 residue and nitrogen of Arg290 residue.
However, this bond is also present in the replacement
of Phe311 residue by Ser or Thr but disappears in
mutants PseFDH-Phe311Asn. The same mutation on
bonds are Arg259 and His312 in CtFDH (Yilmazer et al.
2020). These four residues form a network of hydro-
gen bonds staying tightly in place, thus controlling
the exact positioning of the substrates (Figure 3). The
Asn120Ala mutation possibly disrupts the H-bonding
required for the exact positioning of the substrate at
the enzyme active site, resulting in a loss of formate
oxidation activity. Although there is one additional
hydroxyl group in hydrogen carbonate compared to
formate, the results show that the carbon dioxide
reduction reaction still occurs, albeit with reduced effi-
ciencies after the Asn120Ala mutation. In the
Asn120Ala mutant, the formate molecule would move
away from the catalytic position in the active site
(Figure 3). This situation prevents the exact positioning
of the formate carbon required for the forward reac-
tion transfer of the hydride ion to the cofactor.
However, the results show that the carbon dioxide
molecule makes the reverse reaction less sensitive to
the exact positioning of the substrate carbon.
According to previous studies, mutations in these
positions cause loosening of the tight control in the
substrate positioning, giving flexibility to the conform-
ation of the active site residues (Pala et al. 2018). In
our study, Asn120Ala, a change to a less bulky amino
acid, can cause this effect. The flexibility means that
the hydrogen carbonates can more easily move in
their places. Decreasing of the binding in the mutant
Asn120Ala increased the catalysis rate from 0.25 to
1.73/s like the Asn120Cys mutant (Table 5) (Pala et al.
2018). Stronger binding of hydrogen carbonate could
be the reason for the low catalysis rate by the
wild type.
The Ile94Ala, another mutation in the substrate-
binding site, showed similar effects to Asn120Ala in
the reverse reaction but did not lose its activity in the
forward reaction, although it was dramatically
decreased. The formate molecule in the active site is
kept in place by H-bonds to the main chain nitrogen
of Ile94 (Yilmazer et al. 2020). Although alanine is not
very different from isoleucine, the dramatic increasing
effect of the mutation on KM value (3.7 to 20.8 mM)
indicates that the H-bond between the formate and
the Ala94 is disturbed. Nevertheless, the Ile94Ala
mutant is active, with higher kcat values than the wild
type. In the reverse reaction, the smaller Alanine muta-
tion introduces more space and more conformational
flexibility to the active site as the Asn120Ala mutation
does. Also, poorer bonding of the substrate can
improve product release (Pala et al. 2018), which may
explain higher kcat values.
SoyFDH studied by Alekseeva et al. and Tishkov et al.
Formate
resulted in increased KM
(Table 5). The reason
for this is that Phe is a hydrophobic residue and con-
tains a benzene ring, while Asn is an uncharged amino
acid. It is thought to be that the Phe residue on the
CsFDH-Asn312Phe mutant may cause a slight change
in the folding of the protein, causing new H-bonds to
form, positively affecting the substrate affinity.
In formate oxidation and CO2 reduction, the CsFDH
Val313Pro mutation enhanced the catalysis rate 1.3-
fold and 18.3-fold respectively, while substrate-binding
affinity decreased 1.5-fold and increased 28.4-fold
respectively. Since the Val313Pro mutant had the high-
est catalytic efficiency on CO2 reduction with 0.394/
s.mM, it can be concluded that the hydrophobicity at
this position is crucial for the appropriate orientation
of His312 and accommodation of substrates (Aslan
et al. 2017, C¸akar et al. 2020, Yilmazer et al. 2020).
According to the crystal structure (formate) and
docking information (hydrogen carbonate) of CtFDH,
the substrates were bound in the active site of CtFDH
with H-bonds with Ile94, Asn120, Arg259, His312, and
Gly315 (Ile94-O 2.7 Å and N 3.0 Å, Asn120-ND2 3.0 Å,
R259-NH1 3.1 Å and NH2 3.4 Å, His312-CE1 3.1 Å and
NE1 3.1 Å for formate and Ile94-HN 2.4 Å, Asn120-
2HD2 2.2 Å, Arg259-1HH2 1.6 Å, His312-HE2 2.3 Å and
Gly315-CA 3.5 Å for hydrogen carbonate, Figure 3).
Asn120 and Ile94 are the side chains in hydrogen-
bonding distance from the substrate. They seem to
coordinate the substrate into its place. The other side
chains keeping the substrate in its place by hydrogen

8
K. KOÇDEMIR ET AL.
Table 5. Functional effects of active site mutations in NADþ-dependent formate dehydrogenases.
Mutation
Source
Substrate/Cofactor
Results
Reference
This study
Asn120Ala
CtFDH
Hydrogen carbonate
kcat is increased 6.9-fold, KM is increased
2.7-fold
Asn120Cys
Asn119Tyr
Asn119His
Hydrogen carbonate
Formate
Formate
kcat and KM are increased 7-fold.
KM is decreased 1.4-fold
kcat is decreased 5-fold, KM is decreased
2.2-fold
Pala et al. (2018)
CmFDH
Asn119Asp
Asn119His
Hydrogen carbonate/Formate
Not active with either substrate
KM is increased 1250-fold,
0.1% activity, KM is increased 78-fold
KM is increased 0.8-fold
CboFDH
PseFDH
NADþ
Labrou and Rigden (2001)
Tishkov et al. (1996)
Formate
Asn146Ser
NADþ
Formate
Formate
NADþ
Vmax is decreased 2-fold
KM is increased 60-fold
KM is increased 5.8-fold
KM is decreased 4-fold
kcat is decreased 2-fold, KM is decreased
1.3-fold
Asn146Cys
Asn146Ala
Asn312Phe
CsFDH
Hydrogen carbonate
Formate
This study
Phe311Tyr
Phe311Asn
PseFDH
Formate
KM is decreased 2.1-fold, thermostability is
increased 1.6-fold
KM is increased 4.1-fold,
kcat is decreased 1.4-fold, KM is increased
2.5-fold, thermostability is decreased
2-fold
Tishkov et al. (2015)
NADþ
Formate
Phe311Ser
Phe311Asp
Phe290Ser
NADþ
KM is increased 4.1-fold
Formate
kcat is decreased 1.4-fold, KM is increased
7.1-fold, thermostability is decreased
2-fold
NADþ
KM is increased 5.6-fold
Formate
kcat is decreased 1.8-fold, KM is increased
9.3-fold, thermostability is increased
2.4-fold
SoyFDH
NADþ
KM is decreased 1.5-fold
Alekseeva et al. (2012)
Formate
kcat is increased 1.4-fold, KM is increased
2.7-fold, decrease in thermal stability
KM is increased 3-fold, thermal stability
is increased
kcat is increased 1.8-fold, KM is increased
3.3-fold, thermal stability is
highly increased
KM is decreased 1.5-fold
kcat is increased 1.4-fold, KM is decreased
1.4-fold
Phe290Asn
Phe290Asp
Formate
Formate
Alekseeva et al. (2012)
Kargov et al. (2015)
Phe290Ala
Phe290Tyr
NADþ
Formate
NADþ
KM is decreased 1.2-fold,
kcat is increased 1.2-fold, KM is decreased
1.6-fold
Formate
Phe290Gln
Phe290Glu
Formate
Formate
kcat is increased 1.2-fold, thermal stability is
increased 5-fold
kcat is increased 1.6-fold, and KM is
increased 1.9-fold
Phe290Thr
Phe285Tyr
Val310Asn
Formate
Formate
Hydrogen carbonate
Formate
kcat is increased 1.4-fold
CboFDH
CtFDH
Thermal stability is increased 47-fold
Not active with substrate
kcat is decreased 13.3-fold, KM is decreased
2.3-fold
Slusarczyk et al. (2000)
This study
Val310Thr
Val331Thr
His312Ala
Hydrogen carbonate
Formate
Not active with substrate
kcat is decreased 4.7-fold, KM is increased
1.8-fold
Not active with substrate
kcat is decreased 1.9-fold, KM is decreased
1.4-fold
Not active with substrate
kcat is decreased 18-fold, KM is increased
2-fold
This study
CsFDH
CtFDH
CmFDH
Hydrogen carbonate
Formate
This study
Hydrogen carbonate
Formate
This study
His312Asp
His312Cys
His312Ser
His312Tyr
His312Pse
His312Ala
His312Gln
Formate
KM is decreased 1.3-fold
Pala et al. (2018)
Hydrogen carbonate/Formate
Hydrogen carbonate/Formate
Hydrogen carbonate/Formate
Formate
Not active with either substrate
Not active with either substrate
Not active with either substrate
Lost its activity through formate oxidation
Does not affect the binding of NADþ
0.1% activity, KM is increased 10-fold
PseFDH
CboFDH
Tishkov et al. (1996)
NADþ
Formate
Labrou and Rigden (2001)
Fraction analysis of wild-type and mutant FDHs with SDS-PAGE (Supplementary Figure S1).

BIOCATALYSIS AND BIOTRANSFORMATION
9
Figure 3. CtFDH active site residues are involved in the substrate-binding for formate oxidation and CO2 reduction reactions.
(Wild-type and mutant CtFDHs shown by green and blue sticks, respectively.).
The His312 mutation, one of the key conserved resi-
dues in the active site centre of FDHs, pairs with
Gln288 to form a proton relay system required for
acid–base catalysis. His is the best candidate for this
task for both L- and D-specific dehydrogenase families
at the active site of CtFDH. Since the His312 residue
forms H-bonds with both substrates and silencing this
residue with Ala substitution caused a dramatically
abated KM and kcat values, His312 has to provide elec-
trostatic interaction to achieve catalysis (Galkin et al.
2002). Mutations on this position in FDHs from differ-
ent sources resulted in the loss of activity (Table 5).
CtFDH Val310 and CsFDH Val313 residues (which
are strongly conserved among other FDHs as Thr
(Figure 1), together with Asp283 (Asp280 for CsFDH)
have van der Waals interactions on the side chain of
His312 (His 309 for CsFDH). This key residue has H-
bonds with both formate and hydrogen carbonate
ions to keep the substrates in proper orientations.
The CtFDH Val310Asn mutation dramatically abated
the catalysis rate for formate oxidation. Even though
substrate affinity increased, a 13.3-fold decline in the
catalysis rate resulted in a 5.7-fold decrease in catalytic
efficiency. However, since the kcat value was increased
3.6-fold for CO2 reduction, catalytic efficiency did not
change owing to the regression in substrate bind-
ing affinity.
present study indicates that the effect of mutations
can cause small structural changes which create sig-
nificant differences. Previous studies have shown
that alanine mutation in the active site of CtFDH has
a similar effect. Changing to a less-bulky amino acid
disturbs the H bonding of the substrate and weaker
binding provides flexibility for substrate release,
which causes an increase in the catalytic rate. The
Proline mutation at the 313 position of CsFDH shows
very promising results on CO2 reduction compared to
the literature, even though this mutant is not directly
involved in substrate binding. By this, it was under-
stood that the orientation of the key residues with
the amino acids having hydrophobic side chains is
significant for the FDH catalysis. Results of this study
enlighten the role of catalytic residues of CsFDH and
provide a further understanding of the catalytic reac-
tion mechanism of FDHs. Besides, this mutant
enzyme would increase the possibility of FDH using
for CO2 reduction. FDHs are considered as unfavour-
able for electrochemical reduction of CO2 due to
their low catalytic rate constants (kcat) for CO2 reduc-
tion. However, the obtained mutant in this study and
further engineering of FDHs in the light of this study
can be integrated into electrochemical CO2 reduction
systems. FDHs are dimeric enzymes and can be easily
inactivated depending on the process conditions by
separating their subunits. Since the enzyme stability
can be improved by immobilization to enable
enzymes to be applicable in industrial processes,
immobilization of these enzymes can be beneficial
for reducing CO2 on an industrial scale (Avilova et al.
1985, Bolivar et al. 2007, Mateo et al. 2007,
Fernandez-Lafuente 2009, Velasco-Lozano et al. 2017,
Yildirim et al. 2019). Therefore, immobilized mutant
enzyme might provide development of feasible and
reusable CO2 fixation processes.
Conclusion
In conclusion, we have examined the functional
effects of active site mutations in CtFDH and CsFDH.
These effects were clarified by pH and thermostabil-
ity assay, measuring kinetics parameters (KM, kcat),
and molecular modelling. Obtained results were
compared with structurally equivalent positions
which have been studied in the literature. The

10
K. KOÇDEMIR ET AL.
C¸akar MM, Ruupunen J, Mangas-Sanchez J, Birmingham WR,
Yildirim D, Turunen O, Turner NJ, Valjakka J, Binay B.
Acknowledgements
The authors gratefully acknowledge the help and support of
ETDAM (Enzyme Testing and Consultation Center, Gebze
Technical University, Turkey).
2020.
Engineered
formate
dehydrogenase
from
Chaetomium thermophilum, a promising enzymatic solu-
tion for biotechnical CO2 fixation. Biotechnol Lett. 42(11):
2251–2262.
Choe H, Joo JC, Cho DH, Kim MH, Lee SH, Jung KD, Kim YH.
2014. Efficient CO2-reducing activity of NAD-dependent
formate dehydrogenase from Thiobacillus sp. KNK65MA
for formate production from CO 2 gas. PLoS One. 9(7):
e103111.
Disclosure statement
We have no conflict of interest to declare.
Authors’ contributions
Fernandez-Lafuente R. 2009. Stabilization of multimeric
enzymes: strategies to prevent subunit dissociation.
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Florides GA, Christodoulides P. 2009. Global warming and
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Ustinnikova TB, et al. 2002. Site-directed mutagenesis of
the essential arginine of the formate dehydrogenase
active centre. Biochim Biophys Acta. 1594(1):136–149.
BB has conceived and designed the study. BB, BY, MMC¸ and
YAW have predicted the mutations. KK, FS¸, YAW, BY, MMC¸,
MB, IS, and MCB have carried out mutagenesis and charac-
terization studies. MMC¸ has carried out protein modelling.
BB oversaw the project. The manuscript was written by KK,
FS¸, YAW, BY, MMC¸, ESB and BB.
Funding
This work was partially supported by a grant from the
TUBITAK [Grant number: 119Z155].
€ €
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