Mutational and structural analysis of cobalt-containing nitrilehydratase on substrate and metal binding Akimasa Miyanaga1, Shinya Fushinobu1, Kiyoshi Ito2, Hirofumi Shoun1and Takayoshi W
Trang 1Mutational and structural analysis of cobalt-containing nitrile
hydratase on substrate and metal binding
Akimasa Miyanaga1, Shinya Fushinobu1, Kiyoshi Ito2, Hirofumi Shoun1and Takayoshi Wakagi1
1
Department of Biotechnology, The University of Tokyo, Japan;2Life 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 fromthe
structure of the substrate binding pocket, the wild-type
enzyme showed significantly lower Km and Kivalues 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
is located in the active center The bY68F mutant showed an
elevated Kmvalue and a significantly decreased kcatvalue 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 format 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 a2b2 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 Fromstudies on the structure and function of NHase, a possible reaction model was proposed [2] In this model, imidate is produced as a reaction intermediate before it is converted to an am ide
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) forma 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: asfushi@mail.ecc.u-tokyo.ac.jp
Abbreviations: De, absorption coefficient; ICP-AES, inductive coupled
plasma–atomic emission spectroscopy; Co-type NHase,
cobalt-containing nitrile hydratase; Fe-type NHase, iron-cobalt-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)
Trang 2pocket 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.
Trang 3medium), 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-cyano3-cyano-pyridine, in 100 mM
potassiumphos-phate buffer, pH 7.6 The rate of nitrile hydration was
determined from the increase in absorbance at 25C, using
the absorption coefficients (De) of the corresponding
amides Acrylamide (De225¼ 2.9 mMÆcm)1) and
methacryl-amide (De225¼ 3.2 mMÆcm)1) were measured at 225 nm
Benzamide (De242¼ 5.5 mMÆcm)1) was m easured at
242 nm Nicotinamide (De235¼ 3.2 mMÆcm)1), which is
produced from3-cyanopyridine (De235¼ 0.8 mMÆcm)1),
(De233¼ 2.6 mMÆcm)1), which is produced
from4-cyano-pyridine (De233¼ 0.6 mMÆcm)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 mM for n-butyric acid, 0–50 mM for
propionic acid, and 0–100 lM for benzoic acid) and
two substrate concentrations (0.5 and 5 mM
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
TCGAATTCTGAGAGGAGCTC-3¢ (bF), 5¢-GGTCAT
GCCGCGGCCGCCTTCGTG-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 bF and 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 98C for
2 min, 35 PCR cycles were carried out: each cycle comprised incubation at 98C for 15 s, followed by a 30 s incubation
at 55C, 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 bF and bR primers, or the two megaprimers and the aF and aR 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 NotI and 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 37C, 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 mM isopropyl thio-b-D-galactoside and the metal sources (0.25 mMcobalt chloride, 0.25 mMferric chloride,
or nothing) The cells were then cultured for an additional
16 h All subsequent manipulations were performed at
5C After cell harvesting by centrifugation, the pellet was resuspended in 10 mL of 50 mMTris/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 mM 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 theDPS/MOSFLM [23] or theHKL[24] programsuites
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
Trang 4correction 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 Fo–Fcmap At the final stage of refinement of the
aY114T structure, a cobalt atomwas added to the model,
according to the Fo–Fcmap 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 25C after incubation at different
temperatures for 30 min The Tm was defined as the
temperature at which the activity remaining was 50% of
that without any incubation Figures 2–5 were prepared
using programsXFIT[26] andRASTER3D[27]
Results
Kinetic parameters and inhibitor studies
The kinetic parameters of P thermophila NHase for
acrylonitrile, methacrylonitrile, benzonitrile,
3-cyanopyri-dine and 4-cyanopyri3-cyanopyri-dine, were measured (Table 1) In
general, the enzyme exhibited significantly smaller Km
values for aromatic nitriles than for small aliphatic nitriles
n-Butyric acid competitively inhibited P thermophila NHase, which showed a Kivalue of 1.3 mM This Kivalue
is similar to that of Rhodococcus sp N-771 NHase (1.6 mM) [17] The inhibition by propionic acid and benzoic acid was also competitive, the Ki values being 9.9 mM and 33 lM, 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
4C for 1 year, and the catalytic center did not change,
as confimed by analysis of the crystal structure (data not shown)
Complex structure withn-butyric acid
An NHase crystal soaked in 15 mMn-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 atomof 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˚) fromthe oxygen atomof
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 /K m (s)1Æm M )1 ) 537 ND 0.26 5.8 3.4 Methacrylonitrile
k cat /K m (s)1Æm M )1 ) 2040 ND 5.4 103 15 Benzonitrile
k cat /K m (s)1Æm M )1 ) 6150 ND 32 5290 45 Nicotinonitrile
Isonicotinonitrile
Cobalt content
a Value taken fromreference [19].
Trang 5aCys-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-SO2H [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
Beamline KEK-PF SPring-8 SPring-8 SPring-8
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
Unique observations 57,837 31,332 43,459 30,513
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˚)
No of amino acid residues 429 431 431 431
No of cobalt atoms (occupancy) 1 (1.01) 0 1 (0.71) 1 (0.29)
Average B-factors (A˚2)
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.
Trang 6that coordinated in the native structure Moreover, this
carboxylic oxygen atomwas trapped by three oxygen atoms
of the claw setting (aCys-SO2H111, aSer112, and
aCys-SOH113) We conducted crystallographic refinement of
the complex structure with two alternative states, fixing the
position of the oxygen atomforming 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˚) with an oxygen atom of the side-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 blobs of electron densities were seen in the substrate binding sites of these structures instead of the water molecule present in the native structure, these blobs could not be interpreted (data not shown)
Mutagenic analysis of bTyr68
To evaluate the importance of bTyr68, we replaced it with phenylalanine The kcatand Kmvalues of the bY68F mutant are shown in Table 1 The mutant showed significantly decreased activity compared with the wild-type enzyme The
Km value of the mutant was about 10 times higher, and the kcatvalue 100 times lower, respectively, than those of the wild-type enzyme, when acrylonitrile was used as the substrate The Kivalue for n-butyric acid was also 10 times higher than that of the wild-type enzyme, being
18 mM 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 apoenzyme 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 atominto the model structure.
Trang 7supplemented medium produced a similar apoenzyme
(data not shown)
The crystal structure of the apoenzyme was also
deter-mined at 2.0 A˚ resolution (Table 2) A cobalt atomwas 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 thermophilaNHase, 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 Cc2 atom of aThr109
and the Cc1 atom of aVal136 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 fromthe 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 Kmvalue 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 Kmvalue was similar to that of the wild-type enzyme, when benzonitrile was used
To confirmthe existence of a cobalt atomin 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 Fo–Fcmap, 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 atomwas 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 aT109S m utant in red, and the
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.
Trang 8center 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 atomwas observed at
the catalytic center (Fig 3C) The occupancy and
tempera-ture factor of the cobalt atomwere 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 atomof 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 atomwas 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 Kmvalues 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 chlororaphisB23 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 formand 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 The wild-type enzym e, aT109S mutant, and Fe-type NHase are shown in yellow, red and cyan, respectively The Cc2 atom of aThr109 and the Cc1 atom of aVal136 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 fromreference [19].
Trang 9seemto produce the various substrate preferences among
NHases
Proposed role of bTyr68
The bY68F mutant showed not only an increased Kmvalue,
but also a greatly decreased kcatvalue 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 Km 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
Tmvalue was found to be 10 C lower than that of the
wild-type enzyme (data not shown) On the other hand,
the Kmvalue 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
atomof aSer112 was certainly formed However, the
cysteine cluster region was not open, like that of Fe-type
NHase (Fig 5B), and no iron atom was incorporated when
the mutant was expressed in iron-supplemented medium
A co-expression experiment on the mutant with a Fe-type
NHase activator will confirmthis 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.
References
1 Yamada, H & Kobayashi, M (1996) Nitrile hydratase and its application to industrial production of acrylamide Biosci Biotechnol Biochem 60, 1391–1400.
2 Kobayashi, M & Shimizu, S (1998) Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology Nat Biotechnol 16, 733–736.
3 Ikehata, O., Nishiyama, M., Horinouchi, S & Beppu, T (1989) Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli Eur J Biochem 181, 563–570.
4 Nishiyama, M., Horinouchi, S., Kobayashi, M., Nagasawa, T., Yamada, H & Beppu, T (1991) Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23 J Bacteriol 173, 2465–2472.
5 Kobayashi, M., Nishiyama, M., Nagasawa, T., Horinouchi, S., Beppu, T & Yamada, H (1991) Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1 Biochim Bio-phys Acta 1129, 23–33.
6 Yamaki, T., Oikawa, T., Ito, K & Nakamura, T (1997) Cloning and sequencing of a nitrile hydratase gene from Pseudonocardia thermophila JCM3095 J Ferment Bioeng 83, 474–477.
7 Payne, M.S., Wu, S., Fallon, R.D., Tudor, G., Stieglitz, B., Turner, I.M & Nelson, M.J (1997) A stereoselective cobalt-containing nitrile hydratase Biochemistry 36, 5447–5454.
8 Odaka, M., Fujii, K., Hoshino, M., Noguchi, T., Tsujimura, M., Nagashima, S., Yohda, M., Nagamune, T., Inoue, Y & Endo, I (1997) Activity regulation of photoreactive nitrile hydratase by nitric oxide J Am Chem Soc 119, 3785–3791.
9 Bonnet, D., Artaud, I., Moali, C., Petre, D & Mansuy, D (1997) Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light FEBS Lett 409, 216–220.
10 Endo, I., Odaka, M & Yohda, M (1999) An enzyme controlled
by light: the molecular mechanism of photoreactivity in nitrile hydratase Trends Biotechnol 17, 244–248.
11 Nagasawa, T., Nanba, H., Ryuno, K., Takeuchi, K & Yamada,
H (1987) Nitrile hydratase of Pseudomonas chlororaphis B23 Eur.
J Biochem 162, 691–698.
12 Nagasawa, T., Takeuchi, K & Yamada, H (1991) Character-ization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1 Eur J Bio-chem 196, 581–589.
13 Wieser, M., Takauchi, T., Wada, Y., Yamada, H & Nagasawa, T (1998) Low-molecular-mass nitrile hydratase from Rhodococcus rhodochorous J1: purification, substrate specificity and comparison
Trang 10with the analogous high-molcular-mass enzyme FEMS Microbiol.
Lett 169, 17–22.
14 Huang, W., Jia, J., Cum m ings, J., Nelson, M., Schneider, G &
Lindqvist, Y (1997) Crystal structure of nitrile hydratase reveals a
novel centre in a novel fold Structure 5, 691–699.
15 Nagashima, S., Nakasako, M., Dohmae, N., Tsujishima, M.,
Takio, K., Odaka, M., Yohda, M., Kamiya, N & Endo, I (1998)
Novel non-heme iron center of nitrile hydratase with a claw setting
of oxygen atoms Nat Struct Biol 5, 347–351.
16 Murakami, T., Nojiri, M., Nakayama, H., Odaka, M., Yohda,
M., Dohmae, N., Takio, K., Nagamune, T & Endo, I (2000)
Post-translational modification is essential for catalytic activity of
nitrile hydratase Protein Sci 9, 1024–1030.
17 Piersma, S.R., Nojiri, M., Tsujimura, M., Noguchi, T., Odaka, M.,
Yohda, M., Inoue, Y & Endo, I (2000) Arginine 56 mutation in
the b subunit of nitrile hydratase: importance of hydrogen
bond-ing to the non-heme iron center J Inorg Biochem 80, 283–288.
18 Endo, I., Nojiri, M., Tsujishima, M., Nakasako, M., Nagashima,
S., Yohda, M & Odaka, M (2001) Fe-type nitrile hydratase.
J Inorg Biochem 83, 247–253.
19 Miyanaga, A., Fushinobu, S., Ito, K & Wakagi, T (2001) Crystal
structure of cobalt-containing nitrile hydratase Biochem Biophys.
Res Commun 288, 1169–1174.
20 Nojiri, M., Nakayama, H., Odaka, M., Yohda, M., Takio, K &
Endo, I (2000) Cobalt-substituted Fe-type nitrile hydratase of
Rhodococcus sp N-771 FEBS Lett 465, 173–177.
21 Dixon, M & Webb, E.C (1979) Enzymes, 3rd edn Academic
Press, New York.
22 Sarkar, G & Sommer, S.S (1990) The megaprimer method of
site-directed mutagenesis Biotechniques 8, 404–407.
23 Powell, H.R (1999) The Rossmann Fourier autoindexing
algo-rithmin MOSFLM Acta Crystallogr D 55, 1690–1695.
24 Otwinowski, Z & Minor, W (1997) Processing of X-ray
diffrac-tion data collected in oscilladiffrac-tion mode Methods Enzymol 276,
307–326.
25 Brunger, A.T., Adam s, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T & Warren, G.L (1998) Crystallography & NMR System: a new software suite for macromolecular structure determination Acta Crystal-logr D 54, 905–921.
26 McRee, D.E (1999) XtalView/Xfit – A versatile programfor manipulating atomic coordinates and electron density J Struct Biol 125, 156–165.
27 Merritt, E.A & Murphy, M.E.P (1994) Raster3d, Version 2.0 A programfor photorealistic molecular graphics Acta Crystallogr.
D 50, 869–873.
28 Kopf, M.A., Bonnet, D., Artaud, I., Petre, D & Mansuy, D (1996) Key role of alkanoic acids on the spectral properties, activity, and active-site stability of iron-containing nitrile hydratase from Brevibacterium R312 Eur J Biochem 240, 239–244.
29 Tsujimura, M., Odaka, M., Nakamura, H., Dohmae, N.,
Koshi-no, H., Asami, T., HoshiKoshi-no, M., Takio, K., Yoshida, S., Maeda,
M & Endo, I (2003) A novel inhibitor for Fe-type nitrile hydratase: 2-cyano-2-propyl hydroperoxide J Am Chem Soc.
125, 11532–11538.
30 Wu, S., Fallon, R.D & Payne, M.S (1997) Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstreamprotein Appl Microbiol Biotechnol 48, 704–708.
31 Nojiri, M., Yohda, M., Odaka, M., Matsushita, Y., Tsujishima, M., Yoshida, T., Dohmae, N., Takio, K & Endo, I (1999) Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine J Biochem 125, 696–704.
32 Lu, J., Zheng, Y., Yamagishi, H., Odaka, M., Tujimura, M., Maeda, M & Endo, I (2003) Motif CXCC in nitrile hydratase activator is critical for NHase biogenesis in vivo FEBS Lett 553, 391–196.