Báo cáo khóa học: Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding pdf

Mutational and structural analysis of cobalt-containing nitrile
hydratase on substrate and metal binding
Akimasa Miyanaga
1
, Shinya Fushinobu
1
, Kiyoshi Ito
2
, Hirofumi Shoun
1
and Takayoshi Wakagi
1
1
Department of Biotechnology, The University of Tokyo, Japan;
2
Life Science Laboratory, Mitsui Chemicals Inc., Togo,
Mobara-shi, Chiba, Japan
Mutants of a cobalt-containing nitrile hydratase (NHase,
EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095
involved in substrate binding, catalysis and formation of the
active center were constructed, and their characteristics and
crystal structures were investigated. As expected from the
structure of the substrate binding pocket, the wild-type
enzyme showed significantly lower K
m
and K
i
values for
aromatic substrates and inhibitors, respectively, than alipha-
tic ones. In the crystal structure of a complex with an inhibitor
(n-butyric acid) the hydroxyl group of bTyr68 formed

hydrogen bonds with both n-butyric acid and aSer112, which
islocatedintheactivecenter.ThebY68F mutant showed an
elevated K
m
value and a significantly decreasedk
cat
value. The
apoenzyme, which contains no detectable cobalt atom, was
prepared from Escherichia coli cells grown in medium with-
out cobalt ions. It showed no detectable activity. A disulfide
bond between aCys108 and aCys113 was formed in the
apoenzyme structure. In the highly conserved sequence motif
in the cysteine cluster region, two positions are exclusively
conserved in cobalt-containing or iron-containing nitrile
hydratases. Two mutants (aT109S and aY114T) were con-
structed, each residue being replaced with an iron-containing
one. The aT109S mutant showed similar characteristics to
the wild-type enzyme. However, the aY114T mutant showed
a very low cobalt content and catalytic activity compared
with the wild-type enzyme, and oxidative modifications of
aCys111 and aCys113 residues were not observed. The
aTyr114 residue may be involved in the interaction with the
nitrile hydratase activator protein of P. thermophila.
Keywords: cobalt-containing nitrile hydratase; imidate;
Pseudonocardia thermophila; noncorrin cobalt; claw setting.
Nitrile hydratase (NHase, EC 4.2.1.84) catalyzes the hydra-
tion of nitriles to the corresponding amides [1,2]. NHase has
been used in the industrial production of acrylamide and
nicotinamide from the corresponding nitriles.
NHase is a metalloenzyme that contains iron or cobalt

in its catalytic center. Iron-containing (Fe-type) NHase
contains a nonheme iron ion [3,4], and cobalt-containing
(Co-type) NHase contains a noncorrin cobalt ion [5–7].
NHase consists of a and b subunits, the amino acid
sequences of which do not exhibit homology. In all known
NHases, each subunit has a highly homologous amino acid
sequence. In particular, three cysteine residues and one
serine residue in the cysteine cluster region, which
co-ordinate the iron or cobalt ion of the a subunit, and
two arginine residues of the b subunit, are fully conserved
(Fig. 1). Fe-type NHase shows photoreactivity and binds a
nitric oxide (NO) molecule, whereas Co-type NHase does
not [8–10]. Fe-type NHase preferentially hydrates small
aliphatic nitriles [11], whereas Co-type NHase exhibits a
high affinity for aromatic nitriles [12,13].
The crystal structures of Fe-type NHases, in the active
form at 2.65 A
˚
resolution, and in the NO-bound inactive
state, at 1.7 A
˚
resolution, have been reported [14,15]. Two
cysteine residues, coordinated to the iron ion, are post-
translationally modified to cysteine-sulfinic acid and
cysteine-sulfenic acid, yielding a claw setting structure.
These modifications enable a photoreaction and associ-
ation with NO, and are essential for the catalytic activity
[15–18]. NHase from Pseudonocardia thermophila JCM
3095 is an a
2

b
2
heterotetramer and contains cobalt [6].
Recently, we determined the crystal structure of this
Co-type NHase at 1.8 A
˚
resolution [19]. In this structure,
two cysteine residues, coordinated to the cobalt ion, were
modified and had the claw setting, as in the Fe-type
NHase. From studies on the structure and function of
NHase, a possible reaction model was proposed [2]. In
this model, imidate is produced as a reaction intermediate
beforeitisconvertedtoanamide.
Two arginine residues, bArg52 and bArg157, formed
four hydrogen bonds with the modified oxygen atoms. It is
thought that these bonds also stabilize the claw setting. In
the Fe-type NHase, mutants of the two arginine residues
exhibited sharply reduced stability and enzymatic activity
[17,18].
Of the residues of P. thermophila NHase participating in
the recognition of a substrate, three (bLeu48, bPhe51 and
bTrp72) form a hydrophobic pocket [19]. This hydrophobic
Correspondence to S. Fushinobu, Department of Biotechnology, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,
Japan. Fax: + 81 3 5841 5337, Tel.: + 81 3 5841 5151,
E-mail:
Abbreviations: De, absorption coefficient; ICP-AES, inductive coupled
plasma–atomic emission spectroscopy; Co-type NHase, cobalt-
containing nitrile hydratase; Fe-type NHase, iron-containing
nitrile hydratase; NHase, nitrile hydratase; NO, nitric oxide.

Enzymes: nitrile hydratase (EC 4.2.1.84)
(Received 23 October 2003, revised 17 November 2003,
accepted 25 November 2003)
Eur. J. Biochem. 271, 429–438 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03943.x
pocket is thought to accommodate the alkyl chain or
aromatic ring of a nitrile substrate. bTrp72 of the Co-type
NHase from P. thermophila replaces the tyrosine residue of
Fe-type NHase, and the substrate binding pocket in the
Co-type NHase was larger than that in the Fe-type NHase.
This difference seems to be the cause of the different
substrate preferences of Co-type and Fe-type NHases.
Although the structures of Fe-type NHase and Co-type
NHase are very similar, these NHases specifically bind
their own metals [20]. It is unknown why NHase selects
only a single metal: cobalt or iron. There has only been
one report on metal substitution in NHase [20]. When an
Fe-type NHase from Rhodococcus sp. N-771 was expres-
sed in Escherichia coli (cultured in cobalt-supplemented
Fig. 1. Alignment of the amino acid sequences
of nitrile hydratases (NHases). Three cysteine
residues and one serine residue in the cysteine
cluster region, two conserved arginine resi-
dues, and one conserved tyrosine residue, are
highlighted. Triangles indicate residues
involved in the formation of the substrate
binding pocket. Arrows indicate the residues
that were mutated in this study. Asterisks
indicate completely conserved residues.
P. thermo, Pseudonocardia thermophila JCM
3095; R. rhoJ1L, Rhodococcus rhodochrous J1

low-molecular-mass; P. put, Pseudomon-
as putida NRRL-18668; R. rhoJ1H, Rhodo-
coccus rhodochrous J1 high-molecular-mass;
R. sp. Rhodococcus sp.N-771;P.chlo,Pseu-
domonas chlororaphis B23.
430 A. Miyanaga et al.(Eur. J. Biochem. 271) Ó FEBS 2003
medium), without coexpression of the Fe-type NHase
activator, a cobalt ion was incorporated into the catalytic
center of the NHase. However, the cobalt-substituted
Fe-type NHase showed low enzymatic activity. There
have been no reports on metal substitutions in Co-type
NHase. Co-type NHase contains threonine and tyrosine
in the -C(T/S)LCSC(Y/T)- sequence of the active center,
whereas Fe-type NHase contains serine and threonine
(Fig. 1). The differences in the side-chains, especially at
the threonine/serine position, have been thought to be
important [7]. However, there have been no mutational
studies on these residues. In this article, we report
mutational and structural analysis of the substrate binding
and metal specificity of a Co-type NHase.
Experimental procedures
Kinetic study
NHase activity was determined by measuring the hydration
of acrylonitrile, methacrylonitrile, benzonitrile, 3-cyano-
pyridine, or 4-cyanopyridine, in 100 m
M
potassium phos-
phate buffer, pH 7.6. The rate of nitrile hydration was
determined from the increase in absorbance at 25 °C, using
the absorption coefficients (De) of the corresponding

amides. Acrylamide (De
225
¼ 2.9 m
M
Æcm
)1
) and methacryl-
amide (De
225
¼ 3.2 m
M
Æcm
)1
) were measured at 225 nm.
Benzamide (De
242
¼ 5.5 m
M
Æcm
)1
)wasmeasuredat
242 nm. Nicotinamide (De
235
¼ 3.2 m
M
Æcm
)1
), which is
produced from 3-cyanopyridine (De
235

¼ 0.8 m
M
Æcm
)1
),
was measured at 235 nm. Isonicotinamide
(De
233
¼ 2.6 m
M
Æcm
)1
), which is produced from 4-cyano-
pyridine (De
233
¼ 0.6 m
M
Æcm
)1
), was measured at 233 nm.
At least 10 data points were collected for each substrate.
The inhibition constants for n-butyric acid, propionic
acid and benzoic acid were determined using Dixon plots
[21]. Methacrylonitrile was used as a substrate, the
activity being measured using various inhibitor concen-
trations (0–10 m
M
for n-butyric acid, 0–50 m
M
for

propionic acid, and 0–100 l
M
for benzoic acid) and
two substrate concentrations (0.5 and 5 m
M
metacrylo-
nitrile).
Site-directed mutagenesis
An expression plasmid, pUC18-NHase [19], which contains
the genes for the b and a subunits of NHase and for the
NHase activator, was used for mutagenesis. Site-directed
mutagenesis was carried out by using the megaprimer PCR
method [22]. The primers used were as follows: 5¢-GAGC
TC
GAATTCTGAGAGGAGCTC-3¢ (bF), 5¢-GGTCAT
GCC
GCGGCCGCCTTCGTG-3¢ (bR), 5¢-CACGAAGGCG
GCCGCGGCATG-3¢ (aF), 5¢-GCATGCAAGCTTGCA
TGCCGGTG-3¢ (aR), 5¢-CTCGAGTCGCCGTTCTACT
GGCACTGGATC-3¢ (68f), 5¢-GATCCAGTGCCAGTA
GAACGGCGACTCGAG-3¢ (68r), 5¢-CACGTCGTCGT
GTGCTCGCTCTGCTCCTGC-3¢ (109f), 5¢-GCAGGAG
CAGAGCGAGCACACGACGACGTG-3¢ (109r), 5¢-CTC
TGCTCCTGCACCCCATGGCCGGTGCTG-3¢ (114f), and
5¢-CAGCACCGGCCATGGGGTGCAGGAGCAGAG-3¢
(114r). Restriction enzyme recognition sites are underlined
and mutated residues are shown in bold. The r primer had a
sequence completely complementary to that of the corres-
ponding f primer.
The first PCR amplification was performed with KOD-

plus DNA polymerase (Toyobo, Japan), using the bFand
68r primers, bR and 68f primers, aF and 109r primers, aR
and 109f primers, aF and 114r primers, or aR and 114f
primers. Following an initial denaturation at 98 °Cfor
2 min, 35 PCR cycles were carried out: each cycle comprised
incubation at 98 °C for 15 s, followed by a 30 s incubation
at 55 °C, and a 1 min incubation at 68 °C. The second PCR
was also performed with KOD-plus DNA polymerase,
using the two megaprimers and the bFandbR primers, or
the two megaprimers and the aFandaR primers. The same
PCR program, as described above for the first reaction, was
used. The 0.72 kb DNA fragment and pUC18-NHase were
digested with EcoRI and NotI, and the 1.08 kb DNA
fragment and pUC18-NHase were digested with NotIand
HindIII. The PCR product was ligated to a pUC18 plasmid.
The resulting plasmid was then sequenced to confirm the
presence of the mutation.
Expression and purification of each mutant
and the apoenzyme
The mutants were expressed in E. coli JM109. Cells were
grown at 37 °C, in 1 L of Luria–Bertani broth containing
ampicillin (100 mgÆL
)1
). When the attenuance (D) reached
0.3 at 600 nm, the cells were induced by the addition of
0.1 m
M
isopropyl thio-b-
D
-galactoside and the metal

sources (0.25 m
M
cobalt chloride, 0.25 m
M
ferric chloride,
or nothing). The cells were then cultured for an additional
16 h. All subsequent manipulations were performed at
5 °C. After cell harvesting by centrifugation, the pellet was
resuspended in 10 mL of 50 m
M
Tris/HCl (pH 7.6). From
a cell-free extract prepared by sonication, the protein was
purified by ammonium sulfate precipitation (40–70%) and
anion-exchange chromatographies (DEAE–Sepharose and
MonoQ columns). Each mutant and the apoenzyme were
purified in a soluble form.
Crystallization, data collection and crystallographic
refinement
Crystals of the mutants and apoenzyme were grown under
the same conditions as for the wild-type NHase [19].
Crystals of a complex with n-butyric acid were prepared by
soaking a native crystal in a reservoir solution containing
15 m
M
n-butyric acid for 3 h. Before flash-freezing, the
crystals were equilibrated with the reservoir solution con-
taining 20% (v/v) glycerol. Data were collected using a
CCD camera at the BL6A station of the Photon Factory
(Tsukuba, Japan) and the BL38B2 and BL40B2 stations of
SPring-8 (Harima, Japan), at 100 K. Diffraction images

were indexed, integrated, and scaled using the
DPS/MOSFLM
[23] or the
HKL
[24] program suites.
The crystal structure of Co-type NHase (Protein Data
Bank code 1IRE) was used as the first model. At the first
stage of the crystallographic refinement, the models had the
following removed: the side-chain of the mutated residue
in the aT109S and aY114T structures, and a cobalt atom
and three oxygen atoms in the apoenzyme and aY114T
structures. Several rounds of refinement and model
Ó FEBS 2003 Mutants of Co-type NHase (Eur. J. Biochem. 271) 431
correction were carried out using programs
CNS
[25] and
XFIT
[26]. At the final stage of refinement of the complex
structure, n-butyric acid was added to the model according
to the F
o
–F
c
map. At the final stage of refinement of the
aY114T structure, a cobalt atom was added to the model,
according to the F
o
–F
c
map. The coordinates and structure

factors have been deposited in the Protein Data Bank
(codes: 1UGP, 1UGQ, 1UGR and 1UGS).
Analytical procedures
Protein concentrations were determined by using the
bicinchoninic assay (Pierce Chemical Co.), with BSA as
the standard. The cobalt and iron contents were determined
by ICP-AES (SPS-1200 V; Seiko Instruments, Chiba,
Japan) using sample solutions, and standard solutions of
cobalt and iron (Wako Chemical Co.). The thermostabilities
of the mutants and wild-type enzymes were investigated by
measuring the activity at 25 °C after incubation at different
temperatures for 30 min. The T
m
was defined as the
temperature at which the activity remaining was 50% of
that without any incubation. Figures 2–5 were prepared
using programs
XFIT
[26] and
RASTER
3
D
[27].
Results
Kinetic parameters and inhibitor studies
The kinetic parameters of P. thermophila NHase for
acrylonitrile, methacrylonitrile, benzonitrile, 3-cyanopyri-
dine and 4-cyanopyridine, were measured (Table 1). In
general, the enzyme exhibited significantly smaller K
m

values for aromatic nitriles than for small aliphatic nitriles.
n-Butyric acid competitively inhibited P. thermophila
NHase, which showed a K
i
value of 1.3 m
M
.ThisK
i
value
is similar to that of Rhodococcus sp. N-771 NHase (1.6 m
M
)
[17]. The inhibition by propionic acid and benzoic acid was
also competitive, the K
i
values being 9.9 m
M
and 33 l
M
,
respectively. Therefore, the enzyme exhibited significantly
stronger inhibition by aromatic organic acids than by small
aliphatic organic acids. n-Butyric acid is known not only as
a competitive inhibitor but also as a stabilizer of Fe-type
NHase [11,28]. The activity of Fe-type NHase gradually
decreases under conditions with a lack of n-butyric acid. For
Co-type NHases, a native NHase is more stable. P. thermo-
phila NHase retained complete activity when stored at
4 °C for 1 year, and the catalytic center did not change,
as confimed by analysis of the crystal structure (data not

shown).
Complex structure with
n
-butyric acid
An NHase crystal soaked in 15 m
M
n-butyric acid for 3 h
diffracted up to 1.63 A
˚
(Table 2). The refined structure
obtained using this data set closely resembled the native
structure.
Electron density, similar to n-butyric acid in shape, was
observed in the active site, although it was unclear (Fig. 2).
The electron density of the alkyl end was weak, whereas that
of the carboxylic group was strong. Moreover, two types
of conformations seemed to be present in a mixed state. In
one state (designated as type I), one oxygen atom of the
carboxylic group formed a hydrogen bond (2.90 A
˚
)with
bTyr68. On the other hand, one carboxylic oxygen atom
of another state (type II) appeared to be positioned at a
short distance ( 1.4 A
˚
) from the oxygen atom of
Table 1. Kinetic parameters and cobalt contents of the wild-type and mutant proteins. ND, not determined.
Wild-type Apoenzyme bY68F aT109S aY114T
Acrylonitrile
k

cat
(s
)1
) 1910 <0.1 15.2 621 33.9
K
m
(m
M
) 3.6 ND 58 107 9.9
k
cat
/K
m
(s
)1
Æm
M
)1
) 537 ND 0.26 5.8 3.4
Methacrylonitrile
k
cat
(s
)1
) 1000 <0.1 14.1 482 34.6
K
m
(m
M
) 0.49 ND 2.6 4.7 2.3

k
cat
/K
m
(s
)1
Æm
M
)1
) 2040 ND 5.4 103 15
Benzonitrile
k
cat
(s
)1
) 123 <0.1 7.4 132 4.9
K
m
(m
M
) 0.020 ND 0.23 0.025 0.11
k
cat
/K
m
(s
)1
Æm
M
)1

) 6150 ND 32 5290 45
Nicotinonitrile
k
cat
(s
)1
) 131 – – – –
K
m
(mM) 0.12 – – – –
k
cat
/K
m
(s
)1
Æm
M
)1
) 1090 – – – –
Isonicotinonitrile
k
cat
(s
)1
)90––––
K
m
(m
M

) 0.079 – – – –
k
cat
/K
m
(s
)1
Æm
M
)1
) 1140 – – – –
Cobalt content
Co/ab 0.81
a
<0.01 0.83 0.51 0.035
a
Value taken from reference [19].
432 A. Miyanaga et al.(Eur. J. Biochem. 271) Ó FEBS 2003
aCys-SOH113. It is possible that a covalent bond is formed
between the two oxygen atoms, but the precise chemical
species of this state has yet to be elucidated. In the Fe-type
NHase, 2-cyano-2-propyl hydroperoxide irreversibly inac-
tivates the enzyme, probably by the oxidation of aCys-SOH
to aCys-SO
2
H [29]. Carboxylic oxygen atoms of both types
were directly coordinated to a cobalt ion (2.6 and 2.3 A
˚
in
types I and II, respectively), instead of the water molecule

Table 2. Data collection and refinement statistics.
n-Butyric acid
complex Apoenzyme aT109S aY114T
Data collection statistics
Beam line KEK-PF SPring-8 SPring-8 SPring-8
BL6A BL40B2 BL38B2 BL40B2
Wavelength (A
˚
) 0.978 1.00 1.00 1.00
Space group P3
2
21 P3
2
21 P3
2
21 P3
2
21
Cell constant (A
˚
) a ¼ b ¼ 65.564 a ¼ b ¼ 65.362 a ¼ b ¼ 65.437 a ¼ b ¼ 65.517
c ¼ 184.994 c ¼ 184.099 c ¼ 184.257 c ¼ 183.874
Resolution limit (A
˚
) 1.63 2.0 1.8 2.0
Unique observations 57,837 31,332 43,459 30,513
Completeness (%) 99.0 98.5 99.9 94.7
I/r (highest shell) 10.9 (3.2) 11.1 (4.7) 8.6 (3.0) 14.0 (7.3)
R
merge

(highest shell) (%) 5.4 (24.0) 5.7 (20.1) 5.9 (32.2) 4.7 (14.1)
Refinement statistics
Resolution range (A
˚
) 24.6–1.63 19.4–2.0 19.4–1.8 19.5–2.0
R (highest shell) (%) 18.2 (20.5) 18.4 (18.1) 18.9 (21.2) 18.1 (16.7)
R
free
(highest shell) (%) 19.7 (22.2) 22.0 (23.9) 21.3 (23.1) 22.5 (22.7)
R.m.s. deviations from the native structure (1IRE) (A
˚
)
Ca atoms 0.067 0.126 0.128 0.149
All atoms 0.272 0.138 0.311 0.257
No. of amino acid residues 429 431 431 431
No. of water molecules 397 268 330 280
No. of cobalt atoms (occupancy) 1 (1.01) 0 1 (0.71) 1 (0.29)
No. of n-butyric acid 0.5 + 0.5 0 0 0
Average B-factors (A
˚
2
)
Protein 11.7 19.3 19.4 17.4
Solvent 21.7 24.7 29.4 23.3
n-Butyric acid (type I) 27.0
n-Butyric acid (type II) 26.9
Fig. 2. Complex structure with n-butyric acid. (A) F
o
–F
c

electron density map for the catalytic center of the complex structure with n-butyric acid is
shown at the 3 r contour level, using a stereographic representation. The map was constructed prior to the incorporation of n-butyric acid. The type
I n-butyric acid molecule is shown in light grey and the type II molecule in dark grey. The two hydrogen bonds with bTyr68 are represented by
broken lines in light gray, and the short contact between type II n-butyric acid and aCys-SOH113 by a broken line in dark grey.
Ó FEBS 2003 Mutants of Co-type NHase (Eur. J. Biochem. 271) 433
that coordinated in the native structure. Moreover, this
carboxylic oxygen atom was trapped by three oxygen atoms
of the claw setting (aCys-SO
2
H111, aSer112, and aCys-
SOH113). We conducted crystallographic refinement of
the complex structure with two alternative states, fixing the
position of the oxygen atom forming a short contact in the
type II state. The occupancies of both alternative states were
fixed at 0.5, because the values did not change on the
refinement. Average temperature factors of the butyric acid
molecule in the two states are shown in Table 2. On the
basis of the type I complex structure, we focused on bTyr68
as being the key residue in substrate binding and/or
catalysis. The tyrosine residue at this position is fully
conserved in all NHases (Fig. 1). bTyr68 formed another
hydrogen bond (2.55 A
˚
)withanoxygenatomoftheside-
chain of aSer112 (Fig. 2). This hydrogen bond is also
present in the native NHase [19] and Fe-type NHase
structures [15]. The alkyl group of n-butyric acid was
extended in the direction of the hydrophobic pocket.
bPhe37, bLeu48 and bPhe51 are involved in the hydropho-
bic environment around the alkyl group of n-butyric acid.

We also attempted to prepare co-crystals with other
inhibitors, benzoic acid and propionic acid. The crystals
diffracted up to high resolution (benzoic acid, 1.7 A
˚
;and
propionic acid, 1.5 A
˚
). Although some ÔblobsÕ of electron
densities were seen in the substrate binding sites of these
structures instead of the water molecule present in the native
structure, these ÔblobsÕ could not be interpreted (data not
shown).
Mutagenic analysis of bTyr68
To evaluate the importance of bTyr68, we replaced it with
phenylalanine. The k
cat
and K
m
values of the bY68F mutant
are shown in Table 1. The mutant showed significantly
decreased activity compared with the wild-type enzyme. The
K
m
value of the mutant was about 10 times higher, and
the k
cat
value 100 times lower, respectively, than those of
the wild-type enzyme, when acrylonitrile was used as the
substrate. The K
i

value for n-butyric acid was also  10
times higher than that of the wild-type enzyme, being
18 m
M
.
The crystal structure of the bY68F mutant was deter-
mined at 2.0 A
˚
resolution. The structure of the bY68F
mutant was almost identical to that of the wild-type enzyme,
including the formation of a normal claw setting, except for
the mutation site (data not shown).
Apoenzyme of NHase
The ÔapoenzymeÕ of NHase, which contains no metal ion,
was prepared by expressing the protein in E. coli in a
medium lacking cobalt chloride. The apoenzyme did not
show any detectable enzymatic activity. Moreover,
analysis (by ICP-AES) of the metal content of the
apoenzyme, revealed that it did not contain a cobalt or
an iron ion. Expression of the wild-type enzyme in iron-
Fig. 3. The metal centers of (A) the apoenzyme, and (B) aT109S and (C)
aY114T mutants. (A) 2F
o
–F
c
electron density map at the 2 r contour
level. The disulfide bond is shown by a dotted line. (B) F
o
–F
c

electron
density map (6 r) constructed prior to incorporation of the three
modified oxygen atoms and a cobalt atom into the model structure. (C)
F
o
–F
c
electron density map (6 r) constructed prior to incorporation of
a cobalt atom into the model structure.
434 A. Miyanaga et al.(Eur. J. Biochem. 271) Ó FEBS 2003
supplemented medium produced a similar ÔapoenzymeÕ
(data not shown).
The crystal structure of the apoenzyme was also deter-
mined at 2.0 A
˚
resolution (Table 2). A cobalt atom was not
observed in the catalytic center of the apoenzyme, and
modifications of cysteine residues and the formation of a
claw setting were not observed (Fig. 3A). Instead, electron
density connecting aCys108 and aCys113 was observed,
indicating the formation of a disulfide bond between these
residues. Incubation of the apoenzyme with cobalt ions did
not recover the NHase activity, the incorporation of a
cobalt ion probably being blocked by the disulfide bond.
The conformation of the cysteine cluster region was
significantly closed compared with that of the wild-type
enzyme (Fig. 4).
Structural differences between Co-type and Fe-type
NHases
In the highly conserved cysteine cluster region, two amino

acid residues, corresponding to positions a109 and a114 in
P. thermophila NHase, showed significant conservation in
Co-type and Fe-type NHases. aThr109 and aTyr114 were
conserved in Co-type NHase, whereas, in Fe-type NHase,
these residues were replaced with serine and threonine,
respectively (Fig. 1).
aThr109 is located in the cysteine cluster region. In
Co-type NHase, the side-chain of aThr109 undergoes a
hydrophobic interaction with the side-chain of aVal136
(Fig. 5A). The distance between the Cc2atomofaThr109
and the Cc1atomofaVal136 is 3.7 A
˚
. On the other hand,
in Fe-type NHase, the side-chain of the corresponding
serine residue does not interact with the valine residue.
aTyr114 is also located near the cysteine cluster region.
In Co-type NHase, the hydroxyl group of aTyr114 forms
hydrogen bonds with the main-chain oxygen atoms of
aLeu119 and aLeu121, via a water molecule. Moreover, the
aTyr114 residue undergoes hydrophobic interactions with
its surroundings. In Fe-type NHase, the corresponding
threonine residue (aThr115) forms a hydrogen bond with
the main-chain oxygen atom of a serine residue (aSer113) in
the cysteine cluster (Fig. 5B). The conformation of the
cysteine cluster region of Fe-type NHase is slightly open,
compared with the Co-type NHase (Fig. 5B) [19], although
this difference ( 0.1 A
˚
) was smaller than the coordinate
errors estimated from the Luzzati plot (0.17 A

˚
for 1IRE,
and 0.19 A
˚
for 2AHJ). The hydrogen bond seems to pull
aSer113 closer to aThr115, and thus makes the conforma-
tion of the cysteine cluster region open.
Mutagenic analysis of aThr109
To evaluate the effect of aThr109 on metal selectivity, we
replaced it with serine to produce the aT109S mutant.
Judging from the results of ICP-AES, the aT109S mutant
contained 0.51 cobalt ions per ab heterodimer. The cobalt
content of this mutant was slightly decreased compared with
that of the wild-type enzyme (Table 1). The K
m
value was
30 times higher than that of the wild-type enzyme, when
acrylonitrile was used as the substrate (Table 1). On the
other hand, the K
m
value was similar to that of the wild-type
enzyme, when benzonitrile was used.
To confirm the existence of a cobalt atom in the active
center of the aT109S mutant, the crystal structure of the
aT109S mutant was determined at 1.8 A
˚
resolution
(Table 2). In the F
o
–F

c
map, an electron density peak of a
cobalt atom (10 r), as well as three oxygen atoms of the
modified cysteine residues, were clearly observed (Fig. 3B).
The occupancy of the cobalt atom was found to be 0.71.
Therefore, the structure of this mutant was almost identical
to that of the wild-type enzyme, except for the mutation site.
Mutagenic analysis of aTyr114
To evaluate the effect of aTyr114 on metal selectivity, we
replaced it with threonine to produce the aY114T
mutant. Judging from the results of ICP-AES, the
aY114T mutant contained only 0.035 cobalt ions per
ab heterodimer (Table 1). The mutant exhibited only
slight activity, probably as a result of the low cobalt
content. When the mutant was expressed in medium
without a cobalt ion, or in iron-supplemented medium,
neither the cobalt content nor the catalytic activity was
detectable. The aT109S/aY114T double mutant showed
characteristics similar to those of the aY114T mutant
(data not shown).
The crystal structure of the aY114T mutant was deter-
mined at 2.0 A
˚
resolution (Table 2). The structure of the
aY114T mutant was almost identical to that of the wild-
type enzyme, except for the mutation site and the catalytic
Fig. 4. Superimposition of the metal centers of
the wild-type enzyme, the apoenzyme, and the
aT109S and aY114T mutants. The wild-type
enzyme is shown in yellow, the apoenzyme in

black, the aT109Smutantinred,andthe
aY114T mutant in purple. Purple arrows
indicate the movement of atoms in the
aY114T mutant compared with that observed
in the wild-type enzyme. The disulfide bond of
the apoenzyme is shown as a black dotted line.
Ó FEBS 2003 Mutants of Co-type NHase (Eur. J. Biochem. 271) 435
center. The electron density map around the metal center
was not clear, probably because of the low cobalt ion
content. A low density peak of cobalt atom was observed at
the catalytic center (Fig. 3C). The occupancy and tempera-
ture factor of the cobalt atom were determined to be 0.29
and 24.3, respectively. Modifications of the cysteine residues
aCys111 and aCys113 were not observed (Fig. 3C); how-
ever, no disulfide bond was detected.
The aThr114 residue of the mutant formed a weak
hydrogen bond (3.1 A
˚
) with an oxygen atom of the main-
chain of aSer112, as in the Fe-type NHase (Fig. 5B).
However, the conformation of the cysteine cluster region
was not open, as in the Fe-type NHase, but closed, as found
in the wild-type Co-type NHase (Fig. 5B). The atoms of the
side-chains of aCys111 and aCys113 were moved to the
outside, and the cobalt atom was also slightly moved
(Fig. 4). As a result, the distances between the cobalt atom
and its ligands in the aY114T mutant became greater,
compared the wild-type enzyme (Table 3).
Discussion
Substrate specificity comparison with other NHases

The kinetic parameters of P. thermophila NHase, with
regard to the size of substrates, were similar to those of the
low-molecular-mass Co-type NHase from R. rhodochrous
J1 [13]. Three putative residues that determine the substrate
specificity of P. thermophila NHase, i.e. bLeu48, bPhe51,
and bTrp72, are fully conserved in the low-molecular-mass
Co-type NHase from R. rhodochrous (Fig. 1). In the high-
molecular-mass Co-type NHase from R. rhodochrous [12],
the kinetic parameters for small aliphatic nitriles were
similar to those found for P. thermophila, but the K
m
values
for aromatic nitriles were 1000 times higher. This enzyme
contains tryptophan and serine residues at the positions
corresponding to bLeu48 and bPhe51 of P. thermophila
NHase, respectively. The Fe-type NHase from Pseudo-
monas chlororaphis B23 shows only minimal hydration of
aromatic nitriles as substrates [11]. The bLeu48, bPhe51
and bTrp72 residues of this enzyme are replaced with
valine, valine, and tyrosine, respectively. These three
residues are fully conserved in Fe-type NHases, and form
a narrower substrate-binding pocket [19]. In summary,
differences in the form and size of the hydrophobic pocket
Fig. 5. Superimpositioning of the metal centers
of the wild-type enzyme and mutants of the
cobalt-containing nitrile hydratase (Co-type
NHase) and the iron-containing (Fe-type)
NHase from Rhodococcus sp. N-771. (A)
Vicinity of aThr109 of the Co-type NHase.
Thewild-typeenzyme,aT109S mutant, and

Fe-type NHase are shown in yellow, red and
cyan, respectively. The Cc2atomofaThr109
and the Cc1atomofaVal136 of the Co-type
NHase are connected by a yellow line. (B)
Vicinity of aTyr114 of the Co-type NHase.
The wild-type, aY114T mutant, and Fe-type
NHase are shown in yellow, purple, and cyan,
respectively. The hydrogen bonds in the
aY114T mutant and Fe-type NHase are
shown as purple and cyan lines, respectively.
Table 3. Distances in the metal centers of the wild-type and mutants.
Wild-type
a
Apoenzyme aT109S aY114T
Atoms and distances (A
˚
)
Co-Sc (aCys108) 2.28 – 2.38 2.51
Co-Sc (aCys111) 2.14 – 2.13 2.38
Co-Sc (aCys113) 2.28 – 2.45 2.66
Co-N (aSer112) 2.09 – 2.07 2.15
Co-N (aCys113) 1.96 – 1.92 2.45
Sc (aCys108)-Sc
(aCys111)
3.28 3.27 3.39 3.61
Sc (aCys108)-Sc
(aCys113)
3.16 2.04 3.15 3.98
Sc (aCys111)-Sc
(aCys113)

3.09 4.28 3.26 3.77
a
Values taken from reference [19].
436 A. Miyanaga et al.(Eur. J. Biochem. 271) Ó FEBS 2003
seem to produce the various substrate preferences among
NHases.
Proposed role of bTyr68
The bY68F mutant showed not only an increased K
m
value,
but also a greatly decreased k
cat
value. This indicates that
the hydroxyl group of the bTyr68 residue plays an
important role, not only in substrate binding but also in
catalysis. In the proposed reaction model for NHase [2], an
imidate intermediate is formed during the reaction. Organic
acids, such as n-butyric acid, may inhibit the enzyme as an
analogue of the imidate intermediate. bTyr68 is probably
involved in the stabilization of this intermediate, as well as in
the claw setting, through the hydrogen bond with aSer112
of the claw setting.
Difference in metal center between Co-type
and Fe-type NHases
Payne et al. suggested the importance of the difference
between threonine and serine in the cysteine cluster [7].
However, the mutant at this position (aT109S) had a
normal active center with a certain amount of a cobalt
ion, indicating that the residue is not critical for metal
selectivity. The mutant showed a high K

m
value for a
small substrate, acrylonitrile (Table 2), probably owing to
an increase in the flexibility of the metal center. When the
thermostability of the aT109S mutant was examined, its
T
m
value was found to be  10 °C lower than that of the
wild-type enzyme (data not shown). On the other hand,
the K
m
value for benzonitrile of the aT109S mutant was
similar to that of the wild-type enzyme (Table 2). The
binding affinity of large aromatic substrates seems to
originate mainly from hydrophobic interactions around
the aromatic ring.
In contrast to the aT109S mutant, the cobalt content of
the aY114T mutant was decreased dramatically (Table 1),
and its two cysteine residues were not modified (Fig. 3C).
Therefore, the aTyr114 residue is clearly important for the
formation of the active center of P. thermophila Co-type
NHase. What kind of structural factor causes such variance?
One possibility is that the enzyme is converted to an
Fe-type NHase by this mutation. A hydrogen bond between
the mutated aThr114 residue and the main-chain oxygen
atom of aSer112 was certainly formed. However, the
cysteine cluster region was not open, like that of Fe-type
NHase(Fig.5B),andnoironatomwasincorporatedwhen
the mutant was expressed in iron-supplemented medium.
A co-expression experiment on the mutant with a Fe-type

NHase activator will confirm this hypothesis.
Another possibility is that the aTyr114 residue is involved
in the interaction with the Co-type NHase activator of
P. thermophila. Co-type NHase activators are not homo-
logous with Fe-type NHase activators, and their functions
are believed to be different [30–32]. Although Fe-type
NHase activators show significant similarity to an ATP-
dependent ion transporter, Co-type NHase activators show
a slight similarity to the b subunit of NHase. The Co-type
NHase activator would assist the formation of the active
center during the maturation steps. aTyr114 is located on
the molecular surface, near the active center, in the a subunit
monomer structure. On the other hand, in the active center
of the apoenzyme, a disulfide bond (not cysteine-sulfinic
acid or cysteine-sulfenic acid) was formed through oxida-
tion. In addition to cysteine residues, aCys108 and aCys113,
the aCys111 residue is located in this vicinity. The active
center of NHase seems to form a disulfide bond easily under
oxidative conditions, if the site contains no metal ion. The
metal activator of NHase may incorporate a metal ion to
prevent the formation of a disulfide bond, and may facilitate
the correct oxidative modification of the cysteine residues.
Acknowledgements
We wish to thank the staff of the Photon Factory and SPring-8 for their
assistance with the data collection. This work was supported by the
National Project on Protein Structural and Functional Analysis.
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