Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles

Article abstract of DOI:10.1016/j.bbrc.2021.08.059

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, βLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant βL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the βHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

Full text of DOI:10.1016/j.bbrc.2021.08.059

Biochemical and Biophysical Research Communications 575 (2021) 8e13
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Biochemical and Biophysical Research Communications
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Substrate access tunnel engineering for improving the catalytic
activity of a thermophilic nitrile hydratase toward pyridine and
pyrazine nitriles
,
,
, b, *
Zhongyi Cheng a 1, Shijin Jiang a 1, Zhemin Zhou a
a Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
b Jiangnan University (Rugao) Food Biotechnology Research Institute, Rugao, 226500, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an
exothermic reaction, a NHase with high activity and stability is needed for amide production. However,
the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this
study, through the combination of substrate access tunnel calculation, residue conservative analysis and
site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic
Received 2 August 2021
Received in revised form
18 August 2021
Accepted 20 August 2021
Available online 25 August 2021
NHase from extremophile Caldalkalibacillus thermarum TA2. A1,
a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine ni-
triles. The specific activity of the corresponding mutant L48H towards 3-cyanopyridine, 2-
bLeu48, was semi-rationally identified as
Keywords:
Nitrile hydratase
Pyridine and pyrazine carboxamide
Substrate access tunnel entrance
Semi-rational design
b
cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent
enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine
carboxamides. Further structural analysis demonstrated that the
hydrogen bond with Glu166, which contributes to the expansion of the entrance of substrate access
tunnel and accelerate substrate migration.
bHis48 could form a long-lasting
a
© 2021 Elsevier Inc. All rights reserved.
1. Introduction
hydratase (NHase, EC 4.2.1.84) has emerged as a green and prom-
ising alternative [4].
Pyridine and pyrazine carboxamides are valuable pharmaceu-
tical compounds. For example, nicotinamide is the amide form of
vitamin B3/PP and has been widely used for various skin conditions
such as non-melanoma skin cancers in high risk individuals [1];
Pyrazinamide is an important front-line drug for the treatment of
tuberculosis (TB) by shortening the therapy course as it kills a
population of semi-dormant tubercle bacilli that are not killed by
other TB drugs [2]; Picolinamide is a poly (ADP-ribose) synthetase
(PARP) inhibitor which can be used for multiple indications, espe-
cially for the treatment of cancer [3]. With respect to the high value
of pyridine and pyrazine carboxamides, various methods have been
applied to their industrial scale production, and bio-hydration of
corresponding pyridine and pyrazine nitriles using nitrile
NHase is one of the most representative biocatalysts that have
been successfully industrialized [5]. However, during nitrile bio-
transformation process, the temperature of the reaction mixture
increases and such exothermic reaction could result in the loss of
NHase activity [6]. Besides, the high concentration of nitrile sub-
strates or amide products causes the partial deactivation of NHase
[7]. Currently industrialized NHases from Rhodococcus rhodochrous
J1, both the low-molecular mass NHase (L-NHase) and the high-
molecular mass NHase (H-NHase), as well as NHases from Rhodo-
coccus sp. N-774 and Pseudomonas chlororaphis B23 (the first and
second generation of industrialized acrylamide production strains),
are vulnerable to high catalytic temperature or high concentration
of nitriles/amides [8]. All these drawbacks result in an uncontrol-
lable increase in energy cost to keep the hydration reaction at a
mild condition. Thus, industry still calls for robust NHases with
high catalytic performance under harsh reaction conditions to
make the bio-hydration process greener.
* Corresponding author. Key Laboratory of Industrial Biotechnology (Ministry of
Education), School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
E-mail address: zhmzhou@jiangnan.edu.cn (Z. Zhou).
Since the large-scale bio-production of amides is hindered by
the poor stability and low catalytic activity of NHase, screening
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bbrc.2021.08.059
0006-291X/© 2021 Elsevier Inc. All rights reserved.

Z. Cheng, S. Jiang and Z. Zhou
Biochemical and Biophysical Research Communications 575 (2021) 8e13
microbial collections for new NHases with high stability and ac-
tivity or retrofitting an existed NHase to obtain desired character-
istics has emerged as promising alternative to meet the industrial
requirement during amide production. We have recently discov-
ered and characterized a new thermophilic NHase from Caldalka-
libacillus thermarum TA2. A1 which showed the best
thermostability among the NHases ever reported [9]. Shen et al.
then justified our results and gave insights into the thermostability
mechanism of this NHase [6]. Such thermophilic enzyme provided
an excellent starting point for making itself an ideal candidate in
industrial amide production, and finding the proper way to further
tailor the catalytic activity of this NHase turns out to be the key to
realize its future industrialization.
Various protein engineering approaches such as domain swap-
ping and subunit fusion have been applied to improve the catalytic
activity of NHases, however, the properties of the obtained NHases
are not always satisfactory [10]. Acting as a key role in catalytic
performance of enzymes, substrate access tunnel has attracted
increasing attention in recent years [11]. Tailoring substrate access
tunnels can substantially increase enzymatic activity, selectivity
and stability, which emerged as an effective strategy for protein
engineering [12,13]. The substrate access tunnel of NHase was
column, proteins were eluted using elution buffer (binding buffer
containing 25 mM desthiobiotin, pH 7.4). The active fractions were
collected, concentrated to 500 mL by ultrafiltration, and applied to a
Superdex 200 10/300 GL column (GE Healthcare UK Ltd.).
2.4. Enzymatic assay
The specific activity of NHase was determined by the increase of
the pyridine and pyrazine carboxamide product. The reaction
mixture was the same as that reported in our previous study [17].
The reaction was carried out at 30 ꢀC for 10 min. The amide con-
centration was analyzed by HPLC equipped with a HITACHI C18
reverse phase column (solvent: acetonitrile/water ¼ 1:2 (v/v)). The
detection wavelength was set to 215 nm. One unit (U) of NHase
activity was defined as the amount of enzyme that produced 1 mmol
amide product per minute under the above assay conditions.
The enzyme concentration for the measurement of kinetic pa-
rameters toward 3-cyanopyridine was set to 0.2 mg/mL. The con-
centrations of 3-cyanopyridine were 10, 20, 50, 100, and 200 mM.
The reaction was terminated with 500 mL of acetonitrile after 2 min.
2.5. Thermal denaturation and substrate/product tolerance analysis
located at the interface of the
a and b subunits, and it has been
proved that mutation on the substrate access tunnel residues of
NHase could modulate its selectivity towards various nitrile sub-
strates [14].
In this study, based on the excellent thermostability of Cal.
tNHase, substrate access tunnel engineering was carried out to
further modify the catalytic activity of Cal. tNHase. One gating
residue located near the entrance of the NHase substrate access
The half-life of target enzymes was determined by pre-
incubating enzyme solutions in the absence of a substrate at
60 ꢀC for different durations. The residual enzyme activity was then
measured under standard conditions described above. The half-life
(t1/2) was defined as the time point at which the residual activity of
NHase retained 50% of its original activity.
The determination of the substrate/product tolerance of NHase
was the same as described in our previous study [10].
tunnel,
of Cal. tNHase towards pyridine and pyrazine nitriles. Its corre-
sponding mutant, L48H, showed tremendous increase of catalytic
bLeu48, was found to play key roles in directing the activity
b
2.6. Computational design of NHase
activity towards several pyridine and pyrazine nitrile substrates
compared with its parent enzyme, proving itself a promising
competitor for the industrial production of amides in the near
future.
The 3-dimensional structure of the NHase mutant was con-
structed using the trRosetta online tool [18]. The docking of 3-
cyanopyridine to the active site of WT PtNHase and its mutant
were evaluated using AutoDock [19]. Molecular dynamics (MD)
simulations were performed using NAMD 2.14 with the Charmm27
force field for 50 ns [20]. The proteins were put into a cubic water
box (73 ꢁ 75 ꢁ 88 Å3) with a layer of water of at least 10 Å in each
dimension. A cutoff of 12 Å for non-bonded interactions was
applied. To account for nonstandard amino acids and cobalt III ions
at the active site that cannot fit the Charmm27 force field, our
previously determined parameters were used [14]. The substrate
access tunnel was calculated and analyzed using CAVER analyst 2.0
[21]. The Min. Probe radius was 0.9 Å and the shell depth was 4 Å.
The tunnel starting point was the geometric center of the NHase
active site.
2. Materials and methods
2.1. Strains, plasmids and chemical reagents
The NHase gene from C. thermarum TA2. A1 was synthesized and
cloned to pET-24a plasmid by GENEWIZ Biotech Co., Ltd. (South
Plainfield, NJ, USA). The NHases were heterologously expressed in
Escherichia coli BL21 (DE3). Pyridine and pyrazine nitriles/carbox-
amides were purchased from TCI (Shanghai) Development Co., Ltd.
All other chemicals were obtained commercially.
2.2. Construction of mutants
3. Results and discussion
Site-directed saturation mutagenesis was performed following a
whole plasmid PCR protocol [15]. PrimeSTAR HS DNA Polymerase
from Takara Bio Inc. (Kusatsu, Japan) was used for all PCRs. The PCR
mixture was described previously [16].
3.1. Identification of potential hotspot residues at the substrate
access tunnel
Substrate access tunnel and substrate binding pocket are the
two core regions that direct an enzymatic catalysis process [22].
Substrate should pass through the access tunnel so that the binding
pocket could accommodate the substrate. Besides, both the sub-
strate access tunnel residues and the substrate binding pocket
residues can affect the orientation of substrate at the active site.
Several docking studies have proved that the substrate binding
pocket of NHase can accommodate pyridine nitriles such as 3-
cyanopyridine while only few researches have focused on the in-
fluence of substrate access tunnel on the catalytic activity of NHase
2.3. Expression and purification of enzymes
The cultivation and expression process were the same as
described in our previous study [10]. AKTA purifier (GE Healthcare
UK Ltd., Amersham, UK) was used for enzyme purification. A
StrepTrap HP column (GE Healthcare UK Ltd.) equilibrated with
binding buffer (20 mM Na2HPO4$12H2O, 280 mM NaCl, 6 mM KCl,
pH 7.4) was used for purifying the NHases with a Strep tag at the N
terminus of the
b subunit. After injecting the supernatant into the
9

Z. Cheng, S. Jiang and Z. Zhou
Biochemical and Biophysical Research Communications 575 (2021) 8e13
Fig. 1. Screen of the best mutant at residue
bLeu48 of Cal. tNHase and stability analysis of
bL48H and its parent enzyme. (A) Comparison of catalytic activity of the WT Cal. tNHase
and its 19 mutants at site
bLeu48. (B) SDS-PAGE of the purified WT enzyme and its bL48H mutant. (C) Determination of the half-life time of the enzymes. (D) Relative activity of the
enzymes under the treatment of 1 M 3-cyanopyridine and (E) 2 M nicotinamide.
towards pyridine and pyrazine nitriles [23,24]. In present study, we
mainly focus on the substrate access tunnel to investigate whether
reshaping the substrate access tunnel could influence the binding
and orientation of pyridine and pyrazine substrates inside the
active site and thus improving the catalytic activity of NHase.
To obtain the precise information of the substrate access tunnel
of Cal. tNHase, we firstly attempted to get the crystal structure of
such enzyme, but unfortunately, no high-resolution data were ob-
tained. Therefore, efforts were made to build the structural model
of Cal. tNHase through homologous modeling. According to the PSI-
directing catalytic performance of NHase, for example,
involved in the hydrogen-bonding network around the catalytic
center [27], and Arg52 plays an important role in a subunit ex-
change. Mutation of these residues resulted in a decrease of the
enzymatic activity [28]. As a result, amino acid conservation anal-
ysis was performed using Consurf Sever to exclude the conserved
residues from the twelve pre-selected amino acid sites [29], as well
as to choose the optimal size of the randomization site and mini-
mize the labor-determining screening effort (Fig. S4). The residues
with conservation grades higher that 7 were excluded, resulting the
aGln90 is
b
BLAST search against the PDB database, the
shared 86% sequence identity to that of NHase from Bacillus sp.
RAPc8 (PDB ID: 2DPP) while the subunit of NHase from Bacillus
a
subunit of Cal. tNHase
number of hotspot residues into eight (
aLeu89, bLeu37, bAla41,
b
Gly42, Tyr46, Leu48, Val121 and Val126) (Fig. S2). Thus, these
b
b
b
b
b
eight tunnel-forming residues were selected for the following li-
brary construction.
smithii SC-J05-1 (PDB ID: 1V29) showed the highest sequence
similarity to that of Cal. tNHase with 63.6% identity. The 3D struc-
ture of Cal. tNHase was then constructed by trRosetta and PRO-
CHECK gave 93.6% residues of the created model in allowed regions
in Ramachandran plot [25] (Fig. S1), indicating that the constructed
model was reliable. Subsequently, a 50 ns molecular dynamics
simulation was carried out for energy minimization and structure
optimization of Cal. tNHase.
3.2. Construction of mutagenesis library and screen of NHase
mutatns
To investigate the effect of distinct amino acid side chains at the
selected residue sites on the catalytic activity of Cal. tNHase, site-
saturation mutagenesis was carried out individually at the afore-
mentioned eight positions. 3-Cyanopyridine was used as the sub-
strate for screening positive mutants from the total 152 variants
Several studies have proved that the most likely substrate access
tunnel of NHase locates at the interface of the
a and b subunits
[14,24]. As a result, the corresponding substrate access tunnel of
Cal. tNHase was calculated by CAVER analyst 2.0 (Fig. S2). Through
residue graph analysis which could give information about the
influence of tunnel surrounding residues on the tunnel profile [26],
twelve residues that might potentially affect the tunnel property
(Figs. S5eS11). Clearly, one mutation at residue bLeu48, bL48H, led
to the tremendous increase of catalytic activity of Cal. tNHase
(Fig. 1A), whereas other mutants only showed minor or negative
changes in their activity (Figs. S5eS11). Therefore, the
bL48H
mutant was purified (Fig. 1B), and its specific activity towards
pyridine and pyrazine nitriles, 3-cyanopyridine, 2-cyanopyridine
and cyanopyrazine, were measured (Table 1). The specific activity
were identified (
Tyr46, Leu48,
is reported that conserved residues located at close vicinity to the
NHase active site and subunit interface play key roles in
a
Leu89,
aGln90,
a
Trp117,
bLeu37,
bAla41, bGly42,
b
b
bPhe51,
bArg52,
b
Val121 and
b
Val126) (Fig. S3). It
of the
bL48H mutant towards 3-cyanopyridine was 902.3 U/mg,
a-b
which is 2.4-fold higher than that of the wild-type (WT) Cal. tNHase
10

Z. Cheng, S. Jiang and Z. Zhou
Biochemical and Biophysical Research Communications 575 (2021) 8e13
Table 1
the reason for the increased activity of WT Cal. tNHase in our study.
For the catalysis of 2-cyanopyridine and cyanopyrazine, the
corresponding specific activity of the bL48H mutant was 608.2 U/
mg and 690.5 U/mg, showing a 2.8-fold and 3.1-fold increase than
that of WT Cal. tNHase (217.2 U/mg and 225.9 U/mg), respectively.
The results indicated that a single tunnel-forming residue might
direct NHase activity towards pyridine and pyrazine nitriles.
To get better insight into the catalytic performance of the WT
Enzyme activity of the WT enzyme and its
pyrazine nitrile substrates.
bL48H mutant towards pyridine and
Enzymes
Specific Activity (U$mgꢂ1
)
3-Cyanopyridine
2-Cyanopyridine
Cyanopyrazine
Wild-type
383.8 12.5
902.3 10.2
217.2 11.3
608.2 17.6
225.9 7.8
690.5 13.8
bL48H
Cal. tNHase and its bL48H mutant, kinetic parameters of these two
enzymes towards 3-cyanopyridine was measured. As shown in
Table 2, when the mutation L48H was introduced, the Km value for
catalyzing 3-cyanopyridine was 0.6 mM, half of that of the WT Cal.
tNHase (1.2 mM), indicating that it might be easier for 3-
Table 2
Kinetic parameters of the WT enzyme and its
cyanopyridine.
b
L48H mutant towards 3-
Enzymes
kcat (sꢂ1
)
Km (mM)
kcat/Km (mMꢂ1$sꢂ1
)
cyanopyridine to bind to the bL48H mutant. The kcat of the bL48H
Wild-type
352.1 7.8
833.2 11.7
1.2 0.27
0.6 0.04
293.4
1388.7
mutant was 833.2 sꢂ1, a 2.4-fold increase compared with the bio-
transformation of 3-cyanopyridine by its parent enzyme (352.1
bL48H
s
ꢂ1), and the catalytic efficiency of the
bL48H mutant was 4.7-times
higher than that of the WT enzyme.
(383.8 U/mg). It is worth noting that the specific activity of WT Cal.
tNHase measured in present study is 2.7 times higher than the
activity reported by Shen's group [6]. This is because we co-
3.3. Stability determination of the bL48H mutant
expressed the activator protein of Cal. tNHase with the
a and b
subunit whereas Shen's group expressed the and subunit of Cal.
tNHase without activator. It is known that activator protein plays
key roles in the maturation process of NHase [30], and this might be
a
b
Since stability-activity trade-off of enzymes occurs during their
evolution journey both in nature and laboratory, we speculated that
whether the stability of the
bL48H mutant would decrease
Fig. 2. Structural analysis of Cal. tNHase and its
tNHase and its L48H mutant. (C) Molecular docking of 3-cyanopyridine into the active site of WT Cal. tNHase. (D) Molecular docking of 3-cyanopyridine into the active site of the
L48H mutant. (E) The overall structure of the substrate access tunnel entrance of the WT Cal. tNHase and the entrance-forming residues. (F) The overall structure of the substrate
access tunnel entrance of the L48H mutant and the entrance-forming residues. (G) The exact size and shape of the substrate access tunnel entrance of the WT Cal. tNHase
bL48H mutant. (A) RMSD values for MD simulation of WT Cal. tNHase and its bL48H mutant. (B) The radius of gyration for WT Cal.
b
b
b
calculated by CAVER. (H) The exact size and shape of the substrate access tunnel entrance of the
bL48H mutant calculated by CAVER.
11

Z. Cheng, S. Jiang and Z. Zhou
Biochemical and Biophysical Research Communications 575 (2021) 8e13
tremendously as its catalytic activity has increased sufficiently. The
residual activity towards 3-cyanopyridine of both WT Cal. tNHase
located at close vicinity to the entrance of substrate access tunnel
(Fig. 2E and F). According to the 50 ns MD simulations on both the
and the
more than 50% of its initial activity after incubation at 60 ꢀC for 3 h
whereas the L48H mutant lost half of its activity after 2 h’ heat
treatment (Fig. 1C). However, during the heat treatment process,
the specific activity of the L48H mutant remained higher than that
b
L48H mutant was monitored. The parent enzyme retained
WT enzyme and its
introduced a long-lasting hydrogen bond between the ND1 atom of
His48 and the OE1 atom of Glu166 (Table S1), resulting in a slight
flip of the side chain (Fig. 2F). Such hydrogen bond couldn't be
observed in the WT enzyme (Fig. 2E). The slight flip of the His48
bL48H mutant, the mutation on the b48 residue
b
b
a
b
b
of the WT Cal. tNHase. Besides, it is reported that the NHase
showing the best thermostability is from Bacillus strain. RAPc8 [31].
Such NHase maintained half of its activity after 2.5 h under 50 ꢀC
whereas at 60 ꢀC, it lost half of its activity within 20 min [32],
side chain increased the size of the substrate access tunnel entrance
(Fig. 2G and H), and might make it easier for pyridine and pyrazine
nitriles to migrate through the substrate access tunnel.
showing a much lower thermostability than that of the
bL48H
Declaration of competing interest
The authors declare no conflict of interests.
Acknowledgements
mutant. Moreover, other studies dedicating to tailor the thermo-
stability of existed NHases have not yet obtained competing
counterparts that exhibit better thermostability and catalytic ac-
tivity than the bL48H mutant in present study.
As strong tolerance to nitrile substrates or amide products is one
of the hallmark properties of a well-industrialized NHase, WT Cal.
This work was financially supported by China Postdoctoral Sci-
ence Foundation, China, grant no. 2020M671335 and the Natural
Sciences Foundation of Jiangsu Province (BK20200624).
tNHase and the
tions of 3-cyanopyridine and nicotinamide. After reaction with 1 M
of 3-cyanopyridine for 30 min, the relative activity of the L48H
mutant was 50.2%, whereas that of the WT PtNHase was 62.3%
(Fig. 1D). The relative activity of the L48H mutant after treatment
bL48H mutant were treated with high concentra-
b
Appendix A. Supplementary data
b
with 2 M nicotinamide for 30 min remained at 90.4%, showing no
significant difference compared with that of the WT PtNHase
(91.2%) (Fig. 1E), indicating a remarkable stability towards pyridine
carboxamide.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2021.08.059.
References
3.4. Structural analysis of the bL48H mutant
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To understand the molecular basis of the fine-tuned catalytic
performance, a three-dimensional model of the L48H mutant was
constructed and optimized after a 50 ns MD simulation. Analysis of
Root Mean Square Deviation (RMSD) plots showed that the mean
values of RMSD for the ab subunits of the WT enzyme was 2.04 Å
while in the case of the bL48H mutant, the mean RMSD value was
2.06 Å (Fig. 2A). The radius of gyration (Rg) represents the
compactness of proteins, and proteins with lower Rg show better
thermostability. As shown in Fig. 2B, the Rg value of the
mutant (22.18 Å) is a little bit higher than that of the WT enzyme
(22.12 Å). The RMSD and Rg analysis demonstrated that the L48H
mutant had a close but lower thermostability compared with its
parent enzyme, which is in good agreement with our wet experi-
mental data.
b
bL48H
b
Molecular docking was subsequently carried out to investigate
the substrate-enzyme affinity. Compared with the binding energy
of the WT enzyme (ꢂ3.82 kcal/mol), the
bL48H mutant showed
lower binding energy (ꢂ4.68 kcal/mol) towards 3-cyanopyridine.
Since the coordination of the nitrile cyano group with the metal ion
at the active site is the initial step of the hydration reaction, the
distance between the cobalt ion and the cyano nitrogen is impor-
tant for the catalytic activity of NHase [33]. The distance between
the cobalt ion and the cyano nitrogen of 3-cyanopyridine in the
b
L48H mutant was 2.7 Å (Fig. 2D), clearly shorter than that of the
WT PtNHase (5.0 Å) (Fig. 2C). The docking results were consistent
with the experimentally determined kinetic parameters, indicating
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