Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Article abstract of DOI:10.1007/s00775-015-1273-3

Abstract A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k _cat = 10 ± 2 s^-1) accounts for less than 1 % of the wild-type activity (k _cat = 1100 ± 30 s^-1) while the K _m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k _cat values of 220 ± 40 and 77 ± 13 s^-1, respectively, and K _m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k _cat value of 132 ± 3 s^-1 and a K _m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 ? resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys¹⁰⁴-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

Full text of DOI:10.1007/s00775-015-1273-3

J Biol Inorg Chem (2015) 20:885–894
DOI 10.1007/s00775-015-1273-3
ORIGINAL PAPER
Analyzing the catalytic role of active site residues in the Fe‑type
nitrile hydratase from Comamonas testosteroni Ni1
Salette Martinez1,2,4 · Rui Wu2 · Karoline Krzywda2 · Veronika Opalka2 · Hei Chan2 ·
Dali Liu2,3 · Richard C. Holz1
Received: 14 February 2015 / Accepted: 24 May 2015 / Published online: 16 June 2015
© SBIC 2015
Abstract A strictly conserved active site arginine residue
(αR157) and two histidine residues (αH80 and αH81) located
near the active site of the Fe-type nitrile hydratase from Coma-
monas testosteroni Ni1 (CtNHase), were mutated. These
mutant enzymes were examined for their ability to bind iron
and hydrate acrylonitrile. For the αR157A mutant, the residual
activity (kcat = 10 2 s−1) accounts for less than 1 % of the
wild-type activity (kcat = 1100 30 s−1) while the Km value
of 132 3 s−1 and a Km value of 213 61 mM. These data
indicate that all three residues are catalytically important, but
not essential. X-ray crystal structures of the αH80A/αH81A,
αH80W/αH81W, and αR157A mutant CtNHase enzymes
were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively.
In each mutant enzyme, hydrogen-bonding interactions cru-
cial for the catalytic function of the αCys104-SOH ligand are
disrupted. Disruption of these hydrogen bonding interactions
likely alters the nucleophilicity of the sulfenic acid oxygen and
the Lewis acidity of the active site Fe(III) ion.
is nearly unchanged at 205
10 mM. On the other hand,
mutation of the active site pocket αH80 and αH81 residues
to alanine resulted in enzymes with kcat values of 220 40
and 77
and 179
13 s−1, respectively, and Km values of 187
11
18 mM. The double mutant (αH80A/αH81A)
Keywords Nitrile hydratase · Iron · Hydrolysis ·
X-ray crystallography
was also prepared and provided an enzyme with a kcat value
Introduction
An animated Interactive 3D Complement (I3DC) is available in
Proteopedia at: http://proteopedia.org/w/Journal:JBIC:32
Nitrile hydratases (NHases, EC 4.2.1.84) catalyze the hydra-
tion of nitriles to their corresponding amides under ambient
conditions and physiological pH [1–3]. Industrial processes
used to hydrate nitriles to amides (either acid or base hydra-
tion), are often incompatible with the sensitive structures
of many industrially and synthetically relevant compounds.
Therefore, NHases have attracted substantial interest as
biocatalysts in preparative organic chemistry [4]. NHases
are already used in several industrial applications such as
the synthesis of specialty chemicals and pharmaceuticals
including acrylamide and nicotinamide [5, 6]. A key advan-
tage of NHases is their stereoselectivity, which is of particu-
lar importance in the pharmaceutical arena. Moreover, given
the exquisite reaction specificity, high turnover number,
and benign environmental impact, NHases are becoming
increasingly recognized as a “Green” catalyst for the degra-
dation of environmentally harmful nitriles. However, details
of their reaction mechanism remain poorly understood,
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-015-1273-3) contains supplementary
material, which is available to authorized users.
* Dali Liu
dliu@luc.edu
* Richard C. Holz
richard.holz@marquette.edu
1
Department of Chemistry, Marquette University,
Milwaukee, WI 53201, USA
2
Department of Chemistry and Biochemistry, Loyola
University Chicago, Chicago, IL 60660, USA
3
Department of Chemistry and Biochemistry, Loyola
University Chicago, 1068 W. Sheridan Rd.,
Chicago, IL 60626, USA
4
Present Address: Department of Microbiology and Molecular
Genetics, Michigan State University,
East Lansing, MI 48824, USA
1 3

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J Biol Inorg Chem (2015) 20:885–894
Fig. 1 Active site of wild-type
CtNHase (PDB ID: 4FM4) in
ball-and-stick form. The black
dashed lines represent the
hydrogen bonds
hindering further development and exploitation of both bio-
logical and biomimetic nitrile hydrating catalysts.
resides at the bottom of an open cavity [20]. Analysis of its
crystal structure revealed that two α-subunit histidine residues,
αHis80 and αHis81, and an arginine residue, αArg157, inter-
act with a phosphate ion retained in the active site from the
crystallization solution (Fig. 1). Both histidine residues are
located near the active site within the solvent accessible cavity
X-ray crystallographic studies on NHases indicate that
they are α2β2 heterotetramers that contain either a non-heme
Fe(III) ion (Fe-type) or a non-corrin Co(III) ion (Co-type)
[1, 6, 7]. The metal ion is ligated by three cysteine sulfur
atoms, two backbone amide nitrogens, and a water molecule
[8–11]. Two of the active site cysteine residues are post-trans-
lationally modified to cysteine-sulfinic acid (Cys-SO2H) and
cysteine-sulfenic acid (Cys-SOH) yielding an unusual metal
coordination geometry, termed the “claw-setting” [8, 9].
Recently, X-ray crystal structures of a Co-type NHase from
Pseudonocardia thermophila JCM 3095(PtNHase) bound
by butane boronic acid and phenylboronic acid revealed a
covalent bond between the oxygen atom of the sulfenic acid
ligand and the boron atom of the boronic acid [12]. These
data provided the first experimental evidence that the sulfenic
acid ligand can function as a nucleophile, which was sup-
ported by DFT calculations [13] and QM/MM studies [14].
The prevailing dogma is that both Co- and Fe-type NHase
enzymes require the co-expression of an activator protein to be
soluble and fully active [15–19]. Several open reading frames
(ORFs) have been identified just downstream from the struc-
tural α- and β-subunit genes of NHases, and one of these genes
has been proposed to function as an activator protein [15–19].
However, the Fe-type NHase from Comamonas testosteroni
Ni1 (CtNHase) is unusual among NHases in that it can incor-
porate iron into its active site and properly oxidize the equato-
rial cysteine ligands to Cys104-SOH and Cys102-SO2H in the
absence of an activator protein [20]. The X-ray crystal struc-
ture of CtNHase is similar to other Fe-type NHases except
that the Fe(III) ion in the active site is solvent exposed and
while the arginine residue forms hydrogen bonds with Cys104
-
SOH (in CtNHase this residue is observed as a Cys104-SO2H,
due to the conditions used for the crystallization), which was
recently shown to be capable of functioning as a nucleophile
[20]. Neither histidine residue is conserved among Fe- or Co-
type NHases, while the arginine residue is strictly conserved
among both types of NHases (Fig. 2).
In an effort to understand the roles that αHis80, αHis81,
and αR157 play in enzyme maturation and/or catalysis,
these residues were mutated to αH80A, αH81A, αH80A/
αH81A, αH80W/αH81W, αR157A, and αR157K. Each of
these mutant enzymes were investigated via kinetic, induc-
tively coupled mass spectrometry (ICP-MS), UV–Visible
spectroscopy. In addition, the X-ray crystal structures of
three of the mutant enzymes (αH80A/αH81A, αH80W/
αH81W, and αR157A) were solved. Combination of these
data provides insight into the role of these residues in metal
ion incorporation and catalysis for CtNHase.
Materials and methods
Materials
Acrylonitrile, 2-Amino-2-hydroxymethyl-propane-1,3-diol
hydrochloride (Tris–HCl), and 4-(2-hydroxyethyl)
1 3

J Biol Inorg Chem (2015) 20:885–894
887
Ct
:
:
:
:
:
:
---------------MTDNAVMEQRVDALFVLTKELGLVTDQTVPDYEDALMHDWLPQNGAKLVAKAWTDPVFKA
MSVLIDHAKHTGVPGVPEQAPARDRAWALYEALKSKGAVPDGYVEGWKKTFEEDFTPRKGAELVARAWTDPEFRE
MSVTIDHTTENAAPA---QAAVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGAELVARAWTDPEFRQ
-SVTIDHTTENAAPA---QAPVSDRAWALFRALDGKGLVPDGYVEGWKKTFEEDFSPRRGADLVARAWTDPEFRQ
MTENILRKSDEEIQK-----EITARVKALESMLIEQGILTTSMIDRMAEIYENEVGPHLGAKVVVKAWTDPEFKK
----MSEHVNKYTEY-------EARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRK
:
:
:
:
:
:
60
75
72
71
70
64
Re-TG328-2
Rh-R312
Rh-N771
Pt
Rh/HM-J1
Ct
:
:
:
:
:
:
QLLSEGVAASESLGFSFPKHHKHFVVLENTPELHNVICCSLCSCTAFTIIGMAPDWYKELEYRARIVRQARTVLK
LLLTDGTAAVAQYGWLGPQGEY-IVALEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLE
LLLTDGTAAVAQYGYLGPQGEY-IVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLS
LLLTDGTAAVAQYGYLGPQGEY-IVAVEDTPTLKNVIVCSLCSCTAWPILGLPPTWYKSFEYRARVVREPRKVLS
RLLADGTEACKELGIGGLQGED-MMWVENTDEVHHVVVCTLCSCYPWPVLGLPPNWFKEPQYRSRVVREPRQLLK
WLEEDATAAMASLGYAGEQAHQ-ISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADPRGVLK
:
:
:
:
:
:
135
149
146
145
144
138
Re-TG328-2
Rh-R312
Rh-N771
Pt
Rh/HM-J1
Ct
:
:
:
:
:
:
-EIGLDLPESIDIRVWDTTADTRYMVLPLRPQGTEDWSEAQLATLITQDCLIGVSRLEAPFAALPAPAVALGA
-EMGTTLPADTKIRVVDTTAETRYLVIPVRPEGTEGWTAEQLQEIVTKDCLIGVAVPQVP-------------
-EMGTEIASDIEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAIPQVPTV-----------
-EMGTEIASDIEIRVYDTTAETRYMVLPQRPAGTEGWSQEQLQEIVTKDCLIGVAIPQVPTV-----------
EEFGFEVPPSKEIKVWDSSSEMRFVVLPQRPAGTDGWSEEELATLVTRESMIGVE----PAKAVA--------
RDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEEELTKLVSRDSMIGVSNALTPQEVIV--------
:
:
:
:
:
:
207
208
207
206
205
203
Re-TG328-2
Rh-R312
Rh-N771
Pt
Rh/HM-J1
Fig. 2 Alignment of the amino acid sequences of the α-subunits of
Co-type and Fe-type nitrile hydratases. The conserved metal-binding
motif and the arginine residue which hydrogen bonds to the active
site cysteine-sulfenic acid are highlighted in black. The two non-
conserved histidine residues (αHis80 and αHis81) found in CtNHase
are highlighted in gray. Ct, Comamonas testosteroni Ni1 (Fe-type);
Re-TG3282, Rhodococcus equi TG328-2 (Fe-type); Rh-R312, Rho-
dococcus sp. 312 (Fe-type); Rh-N771, Rhodococcus sp. N771 (Fe-
type); Pt, Pseudonocardia thermophila JCM 3095 (Co-type); Rh/
HM-J1, Rhodococcus rhodochrous J1 high-molecular-mass (Co-type)
piperazine-1-ethanesulfonic acid (HEPES) were obtained
from Sigma-Aldrich. All other materials were purchased at
the highest quality available.
25 °C using a Shimadzu UV-2450 PC spectrophotometer in
a 1 mL quartz cuvette with substrate concentrations rang-
ing from 0 to 700 mM. One unit (U) of NHase activity is
defined as the formation of 1 µmol of acrylamide per min-
ute. To obtain the kinetic parameters, Vmax and Km, the ini-
tial velocities from at least two independent measurements
were fitted to the Hill equation by non-linear regression
using OriginPro 9.0 (OriginLab, Northampton, MA).
Expression and purification of CtNHase mutants
Site-directed mutagenesis was performed using the
QuikChange Lightning Multi Site-Directed Mutagenesis
Kit from Agilent Technologies following the manufac-
turer’s instructions. The expression plasmid pSMCtαβHis
containing the α- and β-His6 genes of CtNHase was used
as the template for mutagenesis with the primers listed in
Table S1. The mutations were confirmed by DNA sequenc-
ing at the University of Chicago Cancer Research Center
DNA sequencing facility. Mutant plasmids were trans-
formed into Escherichia coli BL21(DE3) competent cells
(Agilent Technologies) for expression. Wild-type and
mutant CtNHase proteins were expressed and purified by
immobilized metal affinity chromatography as previously
described for the wild-type protein [20]. Purified protein
samples were analyzed by SDS-PAGE. Protein concentra-
tions were determined using a Coomassie (Bradford) Pro-
tein Assay Kit (Thermo Scientific).
Metal analysis and UV–Visible spectroscopy
The metal content of mutant proteins was determined by
ICP-MS. Purified wild-type and mutant proteins along
with a control of buffer (50 mM Tris–HCl, pH 7.5) con-
taining no protein were treated with concentrated nitric
acid and heated at 70 °C for 1 h. The samples were allowed
to cool to room temperature then diluted to a final concen-
tration of 5 % nitric acid. The metal content was analyzed
at the Water Quality Center in the College of Engineering
at Marquette University. UV–Vis absorption spectra were
recorded on a Shimadzu UV-2450 PC spectrophotometer
in 1 mL quartz cuvettes in 50 mM Tris–HCl, pH 7.5, and
300 mM NaCl at 10 °C.
Kinetic assay
Crystallization of mutant proteins and data collection
The activity of wild-type and mutant proteins were deter-
mined by following the hydration of acrylonitrile in
50 mM Tris–HCl pH 7.5 spectrophotometrically at 225 nm
(∆ε225 = 2.9 mM−1 cm−1). All assays were performed at
Crystals of CtNHase mutant proteins were obtained by the
hanging drop vapor diffusion method using the same condi-
tions as those previously described for the wild-type CtNHase
[20]. Optimized conditions were: 1 µl of CtNHase enzyme
1 3

888
J Biol Inorg Chem (2015) 20:885–894
Table 1 Data collection
and refinement statistics for
the CtNHase mutant crystal
structures
Data set
CtNHase αH80A/αH81A CtNHase αH80W/αH81W CtNHase αR157A
(PDBID: 4ZGJ)
(PDBID: 4ZGE)
(PDBID: 4ZGD)
Space Group
Unit cell dimensions
a = b (Å)
P31
P31
P31
112.0
111.7
111.6
c (Å)
476.0
475.9
474.6
α = β (°)
90
90
90
γ (°)
120
120
120
Resolution (Å)
aRmerge (%)
Rpim (%)
36.57–2.00
14.8 (69.9)
19.0 (32.6)
0.859 (0.473)
3.2 (2.2)
99.7 (99.2)
5.6 (5.5)
2,508,285
67.84–2.80
14.5 (72.4)
8.9 (43.4)
0.989 (0.708)
7.3 (1.9)
99.9 (100)
3.6 (3.7)
585,059
163,400
22.9/18.1
26,101
50.60–2.25
15.6 (72.4)
9.3 (43.2)
0.988 (0.593)
6.9 (2.1)
100 (100)
3.8 (3.7)
1,185,275
313,740
20.3/17.3
28,551
CCI_mean
I/sigma (I)
Completeness (%)
Redundancy
No. total reflections
No. unique reflections 450,084
bRwork/cRfree (%)
25.3/21.9
30,101
4561
No. of atoms
No. of solvent atoms
Average B-factors (Å2)
Overall
445
2995
30.3
28.5
30.8
41.7
23.6
48.4
48.4
48.8
39.1
50.4
0.008
1.16
31.8
30.4
32.4
37.7
32.6
0.007
1.07
Protein main chain
Protein side chain
Solvent
Metal ion
RMSD bond length (Å) 0.002
RMSD bond angles (o) 0.73
Ramachandran
Favored (%)
Allowed (%)
Outlier (%)
96.4
2.6
94.9
3.8
96.7
2.5
1.0
1.3
0.8
The values for the highest resolution bin are in parentheses
RMSD root mean square deviation
ꢀ
ꢀ
a
b
c
ꢀ
Linear Rmerge = Σ Iobs − Iavg /ΣIavg
ꢀ
Rwork = Σ|Fobs − Fcalc|/ΣFobs
Five percent of the reflection data were selected at random as a test set and only these data were used to
calculate Rfree
(15, 20, or 30 mg/ml) in 50 mM HEPES pH 7.0 with an
equal volume of the crystallization reservoir solution (1.08 M
K2HPO4, 0.49 M NaH2PO4 with 25 or 30 % (v/v) glycerol).
The best quality CtNHase crystals were obtained at 20 °C
after ~5 days. For X-ray diffraction data collection, the crys-
tals were flash frozen in liquid nitrogen. Data sets were col-
lected at the SBC 19-ID beamline at the Advanced Photon
Source, Argonne National Laboratory (Argonne, IL, USA).
Monochromatic data collection was conducted at a wave-
length of 0.98 Å using a Quantum 315 CCD detector provid-
ing data sets with resolutions ranging from 2.0 to 2.8 Å.
Structure determination and refinement
X-ray data sets for the αH80A/αH81A, αH80W/αH81W,
and αR157A CtNHase mutant enzymes were indexed, inte-
grated and scaled using HKL3000 software and the statis-
tics revealed that the data were of good quality (Table 1)
[21]. The space group for each was P31 with eight copies
of CtNHase heterodimers in each asymmetric unit result-
ing a total solvent content of ~70 %. Molecular replace-
ment was carried out with wild-type CtNHase as the search
model (PDB ID: 4FM4) [20] using the program Phaser [22]
1 3

J Biol Inorg Chem (2015) 20:885–894
889
Table 2 Kinetic constants for the wild-type and mutant CtNHases
~15-fold faster than previously reported [20] and is in line
with the vast majority of Fe-type NHase enzymes while
the Km value is ~1.4-fold lower [20]. The difference in
the observed kcat value is likely due to the rapid purifica-
tion process employed, which allowed the assay to be per-
formed immediately after the enzyme was purified (both
purification and kinetic measurements were performed in a
time-window of <12 h), since it was found that CtNHase
loses catalytic activity over time [20]. Such a decrease in
catalytic activity has been reported for both Co- and Fe-
type NHases and likely results from the oxidation of the
Cys-SOH ligand to Cys-SO2H when stored under aerobic
conditions and in the absence of butyric acid [27, 28].
Mutation of the strictly conserved arginine residue
(αR157), which forms hydrogen bonds with the active site
kcat (s−1
)
Km (mM)
Wild-type
αH80A
1100 30
220 38
77 13
250 20
187 18
179 11
213 61
αH81A
αH80A/αH81A
αH80W/αH81W
αR157A
132
79
3
4
2
4
232
204
239
7
8
1
10
αR157K
32
Acrylonitrile was used as the substrate
from the CCP4 software suite [23]. Once a solution was
obtained, model building was conducted in COOT [24];
rigid-body refinement and restrained refinement was con-
ducted in refmac5 [25]. To remove model bias and achieve
the best refinement result possible, simulated annealing
refinement, TLS refinement, and ordered solvent identi-
fication were conducted using PHENIX.refine [26] while
model building continued until the lowest Rfree/R values
were achieved. Active site metal ion occupancies were ini-
tially set at one resulting in negative difference electron
αCys104-SOH ligand (this residue is observed as αCys104
-
SO2H in the crystal structure due to the amount of time the
crystals were exposed to air during crystallization), (Fig. 2),
to an alanine (αR157A) resulted in a CtNHase enzyme
that exhibited a kcat value of 10 2 s−1 and a Km value of
205 10 mM (Table 2). The residual activity accounts for
less than 1 % of the wild-type activity while the Km value
is nearly unchanged. Similar results were obtained for the
Fe-type NHase from Rhodococcus sp N-771 (RhNHase)
and the closely related Co-type thiocyanate hydrolase from
Thiobacillus thioparus THI115 (TtSCNase) upon muta-
tion of this arginine residue [29–31]. Therefore, αR157
is important for the hydration of nitriles, however, it is
not strictly required for catalysis. In fact, it was recently
reported that deletion of the entire β-protein for the toyo-
camycin nitrile hydratase from Streptomyces rimosus pro-
vides an enzyme with ~0.3 % of the observed WT enzyme
activity [32]. Based on the crystal structure of wild-type
CtNHase, a hydrogen-bond exists between the N2 atom
of αR157 and the O1 atom of the αCys104-SO2H ligand,
density (F –Fc map) around the metal center (not shown).
o
Metal analyses indicated less than one equivalent of iron
per heterodimer, therefore, the final structural model was
refined with iron ion occupancies and those of the three
coordinating residues (CSA102, S103 and CSA104) set
at 0.8. Positive difference electron density (F –Fc map)
o
around this region was observed (not shown) after refine-
ment, which is likely due to the contribution of residues
102–104 from the 20 % apo-enzyme molecules in the crys-
tal. The structure was refined using PHENIX.refine with
the completed model possessing Rfree/R values provided
in Table 2. Since the P31 space group could be indicative
of twining, the L-test for twining was conducted using
the Xtriage program in PHENIX revealing that the inten-
sity statistics behave as expected, indicating no twining.
Coordinates and structure factors have been deposited in
the Research Collaboratory for Structural Bioinformat-
ics (RCSB) Protein Data Bank (PDB) as entries 4ZGJ
(CtNHase αH80A/αH81A), 4ZGE (CtNHase αH80W/
αH81W) and 4ZGD (CtNHase αR157A).
αCys104-SOH in the active form, (2.9 Å). This αCys104
-
SOH residue has been proposed to function as the nucleo-
phile in catalysis [12], and has been suggested to exist in
its protonated form at pH 7.5 [33]. Mutation of αR157 to
lysine (αR157K), a residue that in principle could provide
a hydrogen bond to the active site sulfenic acid ligand,
resulted in an enzyme with a kcat value of 32 4 s−1 and
a Km value of 240 10. These data suggest that the pro-
tonation state and stability of the active site sulfenic acid
ligand is maintained through hydrogen-bond formation
with αR157 and disruption of this hydrogen-bond likely
result in deprotonation of the active site sulfenic acid ligand
decreasing the ability of the catalytic active site to function.
Mutation of the active site pocket residues, αH80 and
αH81 to alanine, resulted in enzymes with kcat values of
220 40 mM and 77 13 s−1, respectively, and Km val-
ues of 187 11 and 179 18 mM (Table 2). The double
mutant (αH80A/αH81A) was also prepared and provided
Results and discussion
The kinetic constants for wild-type CtNHase and the
CtNHase mutant enzymes were obtained by monitoring
the hydration of acrylonitrile to acrylamide at 225 nm and
25 °C in 50 mM Tris–HCl pH 7.5. The wild-type enzyme
exhibited a kcat value of 1,100
of 250 20 mM (Table 2). The kcat value obtained is
30 s−1 and a Km value
1 3

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J Biol Inorg Chem (2015) 20:885–894
an enzyme with a kcat value of 132 3 s−1and a Km value
of 213
61 mM. Therefore, both αH80 and αH81 are
important for the hydration of nitriles, likely participating
in hydrogen-bond networks since they are located in the
solvent accessible channel; however they are not essential
for catalysis. It was hypothesized that the solvent acces-
sibility of the CtNHase active site, both the large opening
and the axial approach direction, provides the necessary
access for metal ion incorporation without the use of an
activator protein [20]. In an attempt to block the channel
leading to the active site, a double mutant containing tryp-
tophan (αH80W/αH81W) was also prepared. This mutant
CtNHase enzyme exhibited a kcat value of 79 4 s−1 and a
Km value of 232 7. These data indicate that access to the
active site Fe(III) ion is not strongly perturbed but the lack
of the histidine residues decreases the enzymes ability to
hydrate acrylonitrile.
Fig. 3 UV-Visible absorption spectra of wild-type and mutant
CtNHases. Spectra were recorded in 50 mM Tris–HCl, pH 7.5, and
300 mM NaCl
As catalytic activity is dependent on the active site metal
ion, the metal content of wild-type CtNHase and each
mutant enzyme was determined by ICP-MS. Iron content
inhibitor such as butyric acid, (iii), changes in hydrogen-
bonding at or near the active site or (iv) oxidation of αCys-
SOH to αCys-SO2H [1, 36, 37]. On the other hand, blue-
shifts are observed by the addition of substrate and also
upon disruption of the hydrogen-bonding network between
βArg56 and Cys114-SOH and βArg56 and Cys112-SO2H in
the Fe-Type NHase from Rhodococcus sp. N-771 [38–40].
These observed shifts have been attributed to a change in
the π-back-donation from the axial cysteine ligand to the
metal center, in response to perturbations at the active site
[41, 42].
Mutation of αR157 to an alanine would result in loss
of the hydrogen-bond between the nitrogen atom of the
guanidinium moiety of αR157 and the O1 oxygen atom
of αCys104-SO2H (2.9 Å) (Fig. 1). The lack of this hydro-
gen bond likely results in a decrease in negative charge
on the sulfenic acid oxygen as this oxygen has been sug-
gested to be protonated at pH 7.5 [33]. This would result in
an increase in electron donation to the Fe(III) center from
the sulfenic acid sulfur ligand. As a consequence of this
change in π-electron donation from the sulfenic acid sulfur
ligand, the amount of π-electron donation from the axial
thiolate ligand to the Fe(III) ion would likely decrease.
A decrease in π-electron donation from the axial thiolate
ligand has been attributed to observed red shifts of simi-
lar magnitude in the S → Fe(III) LMCT band of several
Fe-type NHase enzymes [41, 42]. In contrast, the αR157K
enzyme could still participate in hydrogen-bonding with
active site sulfenic acid ligand, albeit less efficiently, thus
the observed red shift is smaller in magnitude (Table S2).
While no direct hydrogen bond is observed between αH80
or αH81 and the active site ligands, they do form hydro-
gen bonds with solvent molecules that in turn interact with
active site ligands. Therefore, the observed shifts are likely
of wild-type CtNHase was found to be 1.4
0.2 equiva-
lents of iron per (αβ)2 heterotetramer. These data are in
line with the previously reported value of 1.6 equivalents
of iron per (αβ)2 heterotetramer [20]. ICP-MS analysis of
the single point mutants, αH80A, αH81A, αR157A, and
αR157K revealed that 2.5 0.2, 1.6 0.2, 2.1 0.2, and
1.3
tetramer, respectively. The double mutant’s αH80A/αH81A
and αH80W/αH81W contained 1.5 0.2 and 1.2 0.1
0.1 equivalents of iron is present per (αβ)2 hetero-
equivalents of iron per (αβ)2, respectively. These data
are also consistent with those reported for other Fe-Type
NHases, which typically contain between 1.6 and 2.0 iron
ions per (αβ)2 heterotetramer [34, 35]. These data indicate
that mutating the active site αHis80, αHis81, and αR157
residues have little or no effect on the incorporation of iron
into the active site.
Since metal analysis indicated the Fe(III) is present in
the active sites of each mutant enzyme, the UV–visible
spectrum of each enzyme was recorded (Fig. 3). Fe-type
NHases contain an S → Fe(III) ligand-to-metal-charge-
transfer (LMCT) band at ~700 nm whose position is a
good reporter for electronic perturbations occurring at the
metal center that are the result of primary and/or second-
ary coordination sphere changes [1]. All of the CtNHase
mutants exhibited an S → Fe(III) LMCT band with a λmax
near 700 nm; however, for αH80A, αH81A, αR157A,
and αR157K this absorption band was red-shifted with a
corresponding increase in molar absorptivity (ε) (Table
S2). On the other hand, the αH80A/αH81A and αH80W/
αH81W mutant enzymes exhibited a blue-shift along with
an increase in molar absorptivity (Table S2). The observed
red-shifts in this LMCT band are typically observed when
(i) the pH is increased, (ii), upon interaction with an
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J Biol Inorg Chem (2015) 20:885–894
891
the result of disruption of hydrogen-bonds, hydrogen-bond
was observed trans to the αC99 ligand and interpreted as
a water molecule in the αH80A/αH81A structure (Fig. 5b).
Similar to the wild-type enzyme, the equatorial αC102 and
αC104 ligands are fully oxidized to αCys-SO2H in all three
mutant structures since the crystals were obtained under
aerobic conditions over the course of a week. Interestingly,
the phosphate ion, originally present in the active site of the
wild-type CtNHase enzyme is absent in all three structures.
The hydrogen-bond observed between the N2-atom of
αR157 and the O1-atom of αCys104-SO2H (αCys104-SOH
in the active form) in the wild-type enzyme is measured at
2.9 Å; however, in the αH80A/αH81A mutant, this distance
increases to 3.7 Å (Fig. 5b) while in the αH80W/αH81W
enzyme it increases to 4.1 Å (Fig. 5a), both of which are
outside a typical hydrogen-bond distance. Although αR157
in both the αH80A/αH81A and αH80W/αH81W mutant
enzymes is still capable of forming a hydrogen-bond with
the O1 oxygen atom of αCys104-SO2H (αCys104-SOH in the
active form), substitution of αH80 and αH81 for alanine or
tryptophan residues disrupts loops α74–82 (Fig. 4a, Table
S3) and α152–155 (Fig. 4b, Table S3) resulting in αR157
being pulled away from αCys104-SO2H (αCys104-SOH in
the active form), preventing hydrogen-bond formation.
Based on sulfur K-edge EXAFS and geometry-optimized
DFT calculations, the protonation state of the sulfenic acid
was suggested to be Cys-SOH [33]. Therefore, the loss of
this hydrogen bond between αR157 and the O1-atom of
the sulfenic acid ligand will likely decrease the nucleo-
philicity of the sulfenic oxygen atom, consistent with the
observed blue-shift in the S → Fe(III) LMCT band. Since
the sulfenic acid ligand has been shown to be capable of
functioning as the nucleophile in the catalytic reaction [12],
the loss of the hydrogen bond between the αR157 and the
O1-atom of the sulfenic acid ligand would be expected to
significantly decrease the activity. Therefore, the 10-fold
decrease in kcat observed for both αH80A/αH81A and
αH80W/αH81W mutant enzymes is consistent with these
crystallographic data.
networks, or the oxidation of αCys104-SOH to αCys104
-
SO2H. Oxidation of the sulfenic acid is unlikely given that
these spectra were all recorded immediately after purifica-
tion and the enzyme remained fully active.
To gain insight into the structural perturbations that
occur in and around the CtNHase active site upon the muta-
tion of αH80, αH81, and αR157, three mutant enzymes
were crystallyzed, αH80A/αH81A, αH80W/αH81W,
and αR157A. The X-ray crystal structures of these three
CtNHase enzymes were solved to 2.0, 2.8, and 2.25 Å reso-
lutions, respectively. Overall, the X-ray crystal structures
of all three mutant CtNHase enzymes are similar to the
wild-type structure (Fig. 4a) with the α-carbon root mean
squared deviation (rmsd) for the α-subunit within 0.57 Å
(αH80A/αH81A), 0.55 Å (αH80W/αH81W), and 0.22 Å
(αR157A) and the rsmd for the β-subunit is within 0.23 Å
(αH80A/αH81A), 0.19 Å (αH80W/αH81W), and 0.17 Å
(αR157A). Three pronounced differences are observed
among the α-subunits of all three mutant enzymes. The
first is the orientation of the C-terminus (Fig. 4a), which
may simply not bear functional significance. The second
is a loop regions that contains residues α74–82, while
the third is a loop region containing residues α152–155
(Fig. 4b, Table S3). These two loops regions are located in
the vicinity of the metal center and near the mutation sites.
A loop region in the β-subunit containing residues β92–98
is also slightly perturbed (Fig. 4a). The observed shift in
the α74–82 loop region (Fig. 4b, Table S3) in the mutant
enzymes, contribute to a much wider channel leading to
the active site allowing even greater solvent and substrate
access. In addition, a significant change in the orienta-
tion of two lysine residues located on this loop, αK79 and
αK82, is also observed in the αH80A/αH81A and αH80 W/
αH81 W structures (Figure S1). In both structures, the side
chains of the two lysine residues change conformation as
they flip and point away from the channel compared to the
wild-type structure (Figure S1). Such a change in confor-
mation, likely results in the loss of hydrogen-bonding inter-
actions within the active site cavity. Similar perturbations
in the hydrogen-bonding networks around the active sites
of both Fe- and Co-type NHases have been reported when,
for example, second- or third-shell residues in either the α-
or β-subunit have been mutated, yielding enzymes with low
catalytic activities [36, 43].
Interestingly, besides the loss of the hydrogen bond
between the N2-atom of αR157 and the O1-atom of
αCys104-SO2H (Fig. 5C), the αR157A enzyme has the least
structural change in the overall α-subunit (rmsd of 0.225)
and the α74–82 loop region (rmsd of 0.320). These data
suggest that the >100-fold decrease in the catalytic activity
is due to the loss of the hydrogen-bond between αR157 and
αCys104-SOH ligand, which has been suggested to function
as the nucleophile in the catalytic reaction [12]. That hydro-
gen-bond formation to the active site cysteine ligands is cat-
alytically important, is also reflected in studies done on both
Fe- and Co-type NHases. For example, mutation of αR56 in
the Fe-Type NHase from Rhodococcus erythropolis N771,
which forms a hydrogen bond to both the sulfenic acid and
sulfinic acid residues, resulted in an enzyme that exhibits
The active site Fe(III) ions of the three CtNHase
mutants, αH80A/αH81A, αH80W/αH81W, and αR157A
are similar to that of the wild-type enzyme in that they are
bound by three sulfurs from αC99, αC102, and αC104, and
two backbone amide nitrogens (Fig. 5). No electron den-
sity was observed for an exogenous ligand coordinated to
the metal ion trans to αC99 ligand in either the αH80W/
αH81W or αR157A structures. However, electron density
1 3

892
J Biol Inorg Chem (2015) 20:885–894
Fig. 4 Superposition of
wild-type CtNHase (PDB ID:
4FM4) and mutant structures.
a Superposition of the αβ
heterodimer, the wild-type
is in dark magenta, αH80A/
αH81A mutant is in light green,
αH80W/αH81W is in orange,
and the αR157A mutant is in
light blue. The regions with
pronounced difference between
the wild-type and the mutant
enzymes are indicated with
black arrows. b The two loop
regions located in the α-subunit
exhibiting relatively significant
structural differences are indi-
cated by black arrows
only 1 % of the catalytic activity of the wild-type NHase
[29, 43]. Similarly, mutation of αR170 in the Co-Type
NHase from Pseudomonas putida, which corresponds to the
mutated αR157 in CtNHase, to an aspartate resulted in an
enzyme with a 1.6-fold decrease in the catalytic efficiency
[29, 43]. These data underscore the importance of hydro-
gen-bond formation between second sphere residues and the
active site sulfinic and sulfenic acid ligands in NHases.
Summary
Mutation of αH80, αH81, and αR157 result in CtNHase
enzymes with diminished catalytic activity, indicating that
all three residues are catalytically important but not essen-
tial. In each mutant enzyme, hydrogen-bonding interac-
tions crucial for the catalytic function of the αCys104-SOH
ligand are disrupted. Eliminating these hydrogen bonding
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J Biol Inorg Chem (2015) 20:885–894
893
Fig. 5 Active site structures of
the mutant enzymes in ball-and-
stick form with mutated resi-
dues shown in green. a Active
site of the αH80W/αH81W
mutant with electron density
maps (2Fo – Fc) shown in pink
around αR157 and the mutated
residues, αH80W and αH81W.
b Active site of the αH80A/
αH81A mutant with with
2Fo – Fc electron density map
(pink) around the αR157, the
iron ion, a water molecule, and
the mutated residues, αH80A
and αH81A. c Active site of
the αR157A mutant CtNHase
with 2Fo – Fc electron density
map (pink) around the mutated
residue αR157A, αH80, and
αH81. The black dashed lines
represent hydrogen bonds
interactions likely alters the nucleophilicity of the sulfenic
acid oxygen and the Lewis acidity of the active site Fe(III)
ion. Several studies on both Fe- and Co-Type NHases have
shown the importance of the stability and integrity of the
Cys-SOH ligand for catalytic activity in NHases; for exam-
ple, further oxidation to Cys-SO2H leads to inactivation
[27, 37]. Most recently, in our studies with the Co-Type
NHase from P. thermophila JCM 3095, we showed that
Cys-SOH can function as a nucleophile in the hydration
of nitrile to amides by NHase [12]. Therefore stabiliza-
tion of αCys104-SOH in CtNHase through hydrogen-bond
formation with αR157 is important for a fully functional
NHase enzyme. Second, although not directly involved in
catalysis, the two non-conserved αH80 and αH81 residues
appear to play an important role in maintaining the proper
structure of the α-subunit in and around the active site. For
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J Biol Inorg Chem (2015) 20:885–894
19. Wu S, Fallon RD, Payne MS (1997) Appl Microbiol Biotechnol
48:704–708
example, the αH80A/αH81A and αH80W/αH81W mutant
enzymes exhibited two significant structural changes in two
loop regions of the α-subunit (α74–82 and α152–155) that
affected the hydrogen-bonding interaction between αR157
and αCys104-SOH. Therefore, these studies indicate that a
combination of elements that include, the proper structure
of the active site (i.e. stabilization of active site nucleo-
phile through hydrogen-bonding), proper architecture of
the α-subunit, and outer-sphere residues (e.g. αHis80 and
αHis81), are necessary for NHase to efficiently catalyze
nitrile to amides.
20. Kuhn ML, Martinez S, Gumataotao N, Bornscheuer U, Liu D,
Holz RC (2012) Biochem Biophys Res Commun 424:365–370
21. Z. Otwinowski and W. Minor (1997) In: Charles W. Carter, Jr.
(ed) Methods enzymol. Academic Press, pp. 307–326
22. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Sto-
roni LC, Read RJ (2007) J Appl Crystallogr 40:658–674
23. N. Collaborative Computational Project (1994) Acta Crystallogr
Sect D Biol Crystallogr 50:760–763
24. Emsley P, Cowtan K (2004) Acta Crystallogr Sect D: Biol Crys-
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Acknowledgments This work was supported by the National Sci-
ence Foundation (CHE-1412443, RCH and CHE-1308672, DL). HC
gratefully acknowledges the ACS Project SEED for funding a summer
internship. GM/CA @ APS has been funded in whole or in part with
federal funds from the National Cancer Institute (ACB-12002) and
the National Institute of General Medical Sciences (AGM-12006).
28. Arakawa T, Kawano Y, Katayama Y, Nakayama H, Dohmae N,
Yohda M, Odaka M (2009) J Am Chem Soc 131:14838–14843
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Hori S, Ohtaki A, Noguchi K, Katayama Y, Yohda M, Odaka M
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