Structural Basis for Catalysis and Substrate Specificity of Agrobacterium radiobacter N-Carbamoyl-D-amino Acid Amidohydrolase*
Cheng-Yu Chen
,
Wei-Chun Chiu
,
Jai-Shin Liu
,
Wen-Hwei Hsu
¶ || and
Wen-Ching Wang
¶ ||
From the
Institute of Molecular and Cellular
Biology and Department of Life Science, National Tsing Hua University, Hsinchu
30013, Taiwan and
Institute of Molecular
Biology, National Chung Hsing University, Taichung 402, Taiwan
Received for publication, March 7, 2003
, and in revised form, April 18, 2003.
 |
ABSTRACT
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N-Carbamoyl-D-amino acid amidohydrolase is an
industrial biocatalyst to hydrolyze N-carbamoyl-D-amino
acids for producing valuable D-amino acids. The crystal structure
of N-carbamoyl-D-amino acid amidohydrolase in the
unliganded form exhibits a
-
-
-
fold. To investigate
the roles of Cys172, Asn173, Arg175, and
Arg176 in catalysis, C172A, C172S, N173A, R175A, R176A, R175K, and
R176K mutants were constructed and expressed, respectively. All mutants showed
similar CD spectra and had hardly any detectable activity except for R173A
that retained 5% of relative activity. N173A had a decreased value in
kcat or Km, whereas R175K or
R176K showed high Km and very low
kcat values. Crystal structures of C172A and C172S in its
free form and in complex form with a substrate, along with N173A and R175A,
have been determined. Analysis of these structures shows that the overall
structure maintains its four-layer architecture and that there is limited
conformational change within the binding pocket except for R175A. In the
substrate-bound structure, side chains of Glu47, Lys127,
and C172S cluster together toward the carbamoyl moiety of the substrate, and
those of Asn173, Arg175, and Arg176 interact
with the carboxyl group. These results collectively suggest that a
Cys172-Glu47-Lys127 catalytic triad is
involved in the hydrolysis of the carbamoyl moiety and that Arg175
and Arg176 are crucial in binding to the carboxyl moiety, hence
demonstrating substrate specificity. The common (Glu/Asp)-Lys-Cys triad
observed among N-carbamoyl-D-amino acid amidohydrolase,
NitFhit, and another carbamoylase suggests a conserved and robust platform
during evolution, enabling it to catalyze the reactions toward a specific
nitrile or amide efficiently.
 |
INTRODUCTION
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The enzyme N-carbamoyl-D-amino acid amidohydrolase
(D-NCAase)1
hydrolyzes N-carbamoyl-D-amino acids to D-amino
acids, carbon dioxide, and ammonia
(1). Several microorganisms
produce D-NCAase activity including Agrobacterium
(24),
Arthrobacter (5),
Comamonas (6), and
thermotolent bacteria such as Blastobacter sp. A17p-4
(7) and Pseudomonas
sp. strain KNK003A (8). Despite
low sequence identities among different species, D-NCAases require
a strict D-enantiomer of the N-carbamoyl-amino acid as
their substrate
(57).
D-NCAase has been thus utilized as a biocatalyst in the
pharmaceutical industry to produce valuable D-amino acids because
of the high optical specificity. Currently, a two-enzyme reaction process is
applied that starts with inexpensive substrate, D,L-5
monosubstituted hydantoins, that are synthesized from corresponding aldehydes.
The first step is to hydrolyze the substrate using a D-specific
hydantoinase to produce a D-carbamoyl derivative. The
D-carbamoyl derivative is then converted to the corresponding
D-amino acid including D-phenylglycine and
D-p-hydroxyphenylglycine, the basic building blocks of
-lactam antibiotics by a second enzymatic step catalyzed by
D-NCAase (2,
9).
Crystal structure of D-NCAase reveals a tetramer with 222
symmetry; each monomer shows a four-layer
-
-
-
sandwich fold (10,
11). Site-directed mutagenesis
of His129, His144, and His215 in
D-NCAase suggests strict geometric requirements of these conserved
residues to maintain a stable conformation of a putative catalytic cleft.
Within this pocket, the presumptive active residue, Cys172, is just
located at the bottom (12). A
Cys172-Glu47-Lys127 triad near the floor of
this cavity is thus proposed to participate in catalysis, which is similar to
the Cys177-Asp51-Lys144 site of
N-carbamoylsarcosine amidohydrolase (CSHase)
(11). Interestingly, the Nit
domain of Caenorhabditis elegans NitFhit protein
(13) shows a similar fold with
a presumptive identical C-E-K catalytic triad. Given the structural
information and a global sequence analysis, nitrilases, amidases including
D-NCAase, N-acyltransferases, and presumptive amidases,
are classified as a nitrilase superfamily that comprises a C-E-K catalytic
triad (14). The active
cysteine is postulated to attack a carbon in specific nitrile- or
amide-hydrolysis or amide-condensation reactions, resulting in synthesis of
various natural products. None of the crystal structures of the nitrilase
superfamily, however, had substrates in the active site. The interpretation of
the substrate specificity has thus largely relied on modeling
(10,
11). In D-NCAase, a
number of residues nearby Cys172, particularly Asn173,
Arg175, and Arg176, which are located at the same loop
of a solvent-accessible pocket, are indicated to participate in recognizing a
substrate. Here we report that the crystal structures of the catalytically
inactive D-NCAases, C172A or C172S in its free form and in complex
with a substrate,
N-carbamoyl-D-p-hydroxyphenylglycine (HPG), are
extremely similar and that the mutation of the active Cys172 did
not affect the conformation of the active site. Site-directed mutagenesis
studies of Asn173, Arg175, and Arg176, as
well as crystal structures of N173A and R175A, provide further insight for
substrate binding and catalytic mechanism in D-NCAase and may help
in the future rational design of useful biocatalysts.
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EXPERIMENTAL PROCEDURES
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Site-directed MutagenesisSite-directed mutagenesis was
carried out using a TransformerTM site-directed mutagenesis kit from
Clontech with the pQE-NCA clone as the template according to the
manufacturer's protocol. In brief, the selection primer was designed to change
the XhoI site to SmaI site on the DNA target. The mutagenic
primer was designed to induce a defined mutation into the DNA target of
D-NCAase gene. Plasmid DNA isolated from the recipient strain,
Escherichia coli BMH 71-18 mutS, was digested with
XhoI and transformed into chemically treated competent JM109 E.
coli cells. Mutant plasmids were subjected to DNA sequencing to confirm
the successful mutations.
Expression and Purification of Wild-type and Mutant
EnzymesThe recombinant wild-type and mutant enzymes expressed in
E. coli were isolated as described previously
(15). The purified protein was
analyzed by a SDS-PAGE gel to verify the purity. The protein concentration was
assayed according to the Bradford method
(16) with bovine serum albumin
as a standard.
Enzymatic AssaysThe D-NCAase activity was
assayed by monitoring the release of ammonium product, which could be
colorized using Berthelot reaction to produce blue indophenol (625 nm)
(17). The
Km (mM) and kcat
(min-1) values for wild-type and mutant D-NCAase were
determined from initial velocity data in reactions containing enzyme, 0.1
M sodium phosphate buffer (pH 7.0, 37 °C), 5 mM EDTA
with varying concentration of HPG (110-fold
Km).
Circular Dichroism of Wild-type and Mutant
D-NCAasesCD experiments were performed on an AVIV CD
spectropolarimeter (model 62A DS). All scans were performed between 200 and
260 nm (0.1-cm path length) on solutions containing protein (0.5 mg
ml-1), 10 mM HEPES (pH 7.0), and 1 mM EDTA
and were determined as the average of three scans. To access the thermal
stability of wild-type or mutant D-NCAases, the change of
ellipticity at 222 nm was monitored as the protein sample was heated from 20
to 96 °C with a 2 °C increment. Melting temperature
(Tm) curve was normalized according to the
highest CD signal as 1 and the lowest CD signal as 0.
Tm value was calculated at the temperature with
the CD signal of 0.5.
CrystallizationD-NCAase crystals were obtained
by vapor diffusion in hanging drops by mixing the protein solution (
15 mg
ml-1) with precipitating solution at room temperature as described
previously (15). C172A and
C172S mutants in the presence or absence of HPG (2 mM) were
initially grown as microcrystals with the precipitating condition of 1.20
M lithium sulfate and 0.1 M HEPES buffer at pH 7.0. A
microseeding method was then applied to obtain large single crystals (0.5
x 0.4 x 0.1 mm). Crystals of N173A and R175A were formed directly
within 1 week under 1.02 M and 1.24 mM lithium sulfate
in 0.1 M HEPES buffer at pH 7.0, respectively. For R176A and R176K,
no crystals were obtained. All crystals belong to space group
P21 with cell dimensions (see
Table I) and 4 molecules per
asymmetric unit comparable with that of wild-type D-NCAase
(11).
Data Collection and ProcessingFor data collection, crystals
were transferred to mineral oil for a few minutes and then flash-frozen in a
liquid nitrogen stream. C172A crystal data were collected at -150 °C using
a MSC X-Stream Cryo-system with a double-mirror-focused CuK
x-ray
radiation generated from a Rigaku RU-300 rotating anode generator at
Macromolecular x-ray Crystallographic Laboratory of National Tsing Hua
University, Hsinchu, Taiwan. C172S, C172A·HPG, and C172S·HPG
crystal data were collected on beamline 6A at Photon Factory, Tsukuba, Japan
using an ADSC Quantum 4R CCD detector. Each data set was processed and scaled
with MOSFLM (18) and the CCP4
program suites (19). R175A and
N173A crystal data were collected on BL12B2 Taiwan beamline at Spring-8, Sayo,
Japan using an ADSC Quantum 4R CCD detector. Data were processed with the
HKL/HKL2000 suite (20). The
statistics of the data collections are given in
Table I.
Structure Determination and RefinementThe wild-type crystal
model omitting solvent molecules (Protein Data Bank code 1FO6
[PDB]
) was used to
calculate a difference Fourier map with the coefficients
2Fo - Fc and
calculated phases for each mutant or mutant-substrate complex. A tetramer with
the
/
fold was seen for each mutant or mutant-substrate complex.
Clearly visible density for the substituted side chain in a mutant or that for
the bound substrate was observed. A model was thus readily built for each
mutant or mutant-substrate complex using the program O version 8.0
(21).
Structure refinement was carried out with the REFMAC5 program
(22). The four molecules of
the asymmetric unit were refined independently first by restrained refinement
procedure using the maximum-likelihood function. Five percent of the
reflections were randomly selected and used to compute a free R value
(Rfree) for cross-validation of the model. Sigma
A-weighted 2Fo - Fc
and Fo - Fc electron
density maps were generated after each cycle of refinement step. The maps were
then inspected to modify the model manually on an interactive graphics work
station with the program O. The progress of the refinement was evaluated by
the improvement in the quality of the maps, as well as the reduced values for
R and Rfree. Non-crystallographic symmetry
restraints, as well as geometrical restraints, were then applied and gradually
relaxed during the refinement. A cis-peptide between Met73
and Pro74 in each mutant and a sulfate molecule with strong density
in C172A or C172S were then manually built into the model. Coupled with
ARP/wARP program (23), water
molecules were introduced automatically into the model. TLS refinement
(24) prior to individual
isotropic B value refinement was used to further reduce the R and
Rfree values. The stereochemistry of the protein model was
assessed using the program PROCHECK
(25). Estimates of the
coordinate errors were made using the method of Read
(26). A summary of data
collection and the refinement statistics is shown in
Table I.
Structure comparisons among wild-type D-NCAase, mutant
D-NCAase, and mutant-substrate complex structures were carried out
with the program LSQMAN (27)
by superimposing overall C
atoms of a monomer. For binding site
comparison, C
atoms or side-chain atoms of 12 residues surrounding the
binding pocket (Glu47, Lys127, His144,
Glu146, Cys172, Asn173, Arg175,
Arg176, Asn197, Thr198, His201,
and Asn202) were superimposed. A comparison of D-NCAase
with the Nit domain of NitFhit (Protein Data Bank code 1EMS
[PDB]
) or CSHase
(Protein Data Bank code 1NBA
[PDB]
) was done by superimposing side chains of three
catalytic residues (Glu47, Lys127, and Cys172
in D-NCAase; Glu54, Lys127, and
Cys169 in Nit; Asp51, Lys144, and
Cys177 in CSHase). The pictures of three-dimensional structure
models were prepared with MOLSCRIPT
(28) coupled to RASTER3D
(29) programs. The figures of
electron density map were prepared with PyMOL
(www.pymol.org).
 |
RESULTS AND DISCUSSION
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Expression and Enzymatic Analysis of D-NCAase
Mutants Based on the D-NCAase·HPG model
(11), Cys172,
Asn173, Arg175, and Arg176 located in a short
loop near the floor of the binding pocket were chosen for mutational analysis.
Cys172 was replaced with alanine or serine and expressed in E.
coli, respectively. After purification by affinity chromatography, a
major band of an apparent molecular mass of
36 kDa was observed on an
SDS-PAGE gel for each mutant (Fig.
1). Approximately 10 mg of pure C172A protein and 5 mg of pure
C172S protein per liter harvest were obtained, respectively. Enzymatic assay
showed greatly reduced activity for both mutants; there was less than 0.1% of
relative activity for C172S and no detectable activity for C172A. N173A,
R175A, and R176A mutants were then constructed, expressed, and purified,
respectively (Fig. 1). Both
R175A and R176A showed no detectable activity, whereas there was less than 5%
of relative activity for N173A as compared with that of the wild-type enzyme.
We further generated R175K and R176K mutants. For either one, there was less
than 0.1% of relative enzymatic activity.

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FIG. 1. SDS-PAGE analysis of purified D-NCAase mutants. Lane 1,
wild-type D-NCAase; lane 2, C172S; lane 3 C172A;
lane 4, R175A; lane 5, R175K; lane 6, R176A;
lane 7, R176K; lane 7, N173A.
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N173A, R175K, and R176K were subjected for kinetic analysis. As shown in
Table II, R175K and R176K had
2.5- and 4-fold higher Km value,
respectively, as compared with that of wild-type
(Table II). Moreover, the
kcat value was significantly reduced for either of two,
resulting in an extremely lower
kcat/Km value than that of
wild-type. The N173A mutant had
13-fold reduced kcat
but 2.5-fold lower Km.
CD Spectroscopy of Wild-type and Mutant
D-NCAasesCD studies were performed to assess the
conformational integrity and thermal stability for wild-type, C172S, N173A,
R175K, R176A, and R176K. All mutants exhibited far ultraviolet CD spectra
nearly identical to that of wild-type D-NCAase (data not shown),
indicating a similar secondary structure. To compare the stability of the
wild-type and mutant proteins, the unfolding of the protein was then monitored
by the change in ellipticity at 222 nm as the temperature of the sample was
increased. All transitions were found to be cooperative and irreversible and
had comparable thermal stabilities with Tm of 63
to 71 °C (Table II). These
results suggest that each of the created mutants did not affect the secondary
structure, as well as the thermal stability, of the protein.
Crystal Structures of C172A, C172S, R175A, N173A,
C172A·HPG, and C172S·HPGThe
crystal structure of C172S was determined to 2.2 Å by molecular
replacement method. Residues 3304 were continuous and defined well in
the electron density map. The final model was refined to an R of
18.8% (Rfree = 26.7%)
(Table I). Similarly, the
structure of C172A was determined and refined to 2.0 Å resolution, with
an R of 17.9% (Rfree = 23.5%). Crystals of R175A
and N173A were obtained under a similar crystallization condition as that for
wild-type enzyme. Structures were then determined at 2.0 Å (R =
19.0%, Rfree = 24.6%) and 1.95 Å (R =
15.5%, Rfree = 20.9%) for R175A and N173A, respectively.
Estimated coordinate error values are given in
Table I. As shown in
Fig. 2, the substituted
side-chain electron density in residue 172 was clearly visible for either
C172S (Fig. 2A) or
C172A (Fig. 2B). Each
of these mutant structures shows four subunits (ABCD) with 222 symmetry and is
best described as a dimer of dimers like that of the wild-type structure.
Moreover, the monomeric subunit of each mutant demonstrates the wild-type
-
-
-
architecture with modest deviation in the
overall C
atoms (Table
III).

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FIG. 2. The 2Fo -
Fc electron density map of
D-NCAase mutant around residue 172. A, C172S mutant.
B, C172A mutant. Maps are contoured at the 1.5- level.
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TABLE III Comparison of the D-NCAase monomer and binding-site
region Comparison of root mean square deviations (Å) for the
overall C atoms in monomer A, and the C atoms or all atoms of
the binding-site region, between wild-type and mutant, wild-type and
mutant-substrate complex, or the free and the bound structures.
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The C172A·HPG and C172S·HPG structures were determined and
refined to an R of 17.5% (Rfree = 23.3%) and
18.6% (Rfree = 26.5%), respectively
(Table I). As seen in
Fig. 3A, the
2Fo - Fc map
unambiguously identified the location and orientation of the substrate in
either complex structure. The model consists of four subunits (ABCD) and four
substrate molecules bound to the catalytic site of each subunit
(Fig. 3B). Like the
free-form structure, the monomer has a
/
-type structure with two
central
sheets and two helices packed on either side. The four
substrates are located in a solvent-accessible cleft
(Fig. 3C) near the
interface of the compact dimers AB and CD, where a long C-terminal
fragment extends from a helix to a site near a dyad axis and associates with
another monomer. The root mean square deviation in the overall C
atoms
between the superimposed structures with or without substrate is 0.228 Å
for C172A and 0.327 Å for C172S, thus indicating limited conformational
change in the overall structure upon substrate binding
(Table III).

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FIG. 3. Crystal structure of the C172S-substrate complex. A, the
2Fo - Fc map of the
C172S·HPG complex around HPG, contoured at the 1.5- level.
B, ribbon representation of the homotetrameric structure of the
complex, ABCD. The four subunits, A, B, C, and D, are depicted in blue,
yellow, red, and green, respectively. HPG is drawn as a
ball-and-stick model. C, subunit A of C172S with the bound
substrate.
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The Binding PocketThe substrate is bound in a pocket
surrounded by three large loops (4661, 127146, and
197206) and a short loop (172178). A number of residues from
those loops including Glu47, Lys127, His144,
Glu146, Ala172/Ser172, Asn173,
Arg175, Arg176, Asn197, Thr198,
His201, and Asn202 interact with HPG, particularly with
the carbamoyl and the carboxyl moieties (≤3.8 Å)
(Fig. 4A).
Superposition of the C
atoms of the binding site region between the
wild-type and mutant structures shows virtually identical conformation (C172A,
0.150 Å; C172S, 0.147 Å), indicating that substitution of cysteine
with serine or alanine in residue 172 did not perturb the structure of the
binding pocket (Table III).
Likewise, the comparison of the free form with the substrate-bound form showed
very limited change (see Table
III and Fig.
4B), suggesting a sturdy site for substrate binding. In
the free form of either C172A or C172S structure, a sulfate ion is bound near
residue 172 (Fig. 2). Its O2
atom (Ser172 (O
)-sulfate (O2), 2.81 Å) is found in the
nearly equivalent position that is occupied by an O9 atom in the carboxyl
group of the HPG molecule (Ser172 (O
)-HPG (O9), 2.86
Å) (Fig. 3A).
The O atom from a sulfate ion molecule thus interacts with -OH of
Ser172 in the same manner as the carboxyl group of HPG in the
substrate-bound form.

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FIG. 4. The binding pocket of the C172S·HPG complex. A,
stereoview of the C172S·HPG binding pocket. HPG is in yellow.
Four loops enclosing HPG are blue. Glu47,
Lys127, and Ser172 are in red, whereas the
other residues binding to substrate are in green. B, superimposed
structures between C172S and C172S·HPG complex. The protein backbones
of C172S and C172S·HPG are in green and red,
respectively. HPG is in yellow. Glu47, Lys127,
and Ser172 residues of C172S (green) and C172S·HPG
(red) are shown by stick structures. The oxygen and nitrogen atoms
are red and blue, respectively. C, schematic
diagram of HPG bound to C172S. Interactions are shown by dotted
lines. The numbering of HPG is in red.
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There are 11 interactions (≤3.8 Å) between the carboxyl moiety of
HPG and the binding pocket (Ser172, Asn173,
Arg175, Arg176, Asn197, and
Thr198) in the C172S·HPG complex
(Fig. 4C). Among
these, five hydrogen bonds are found: Ser172 (O
)-HPG (O9),
2.86 Å, Asn173 (N
2)-HPG (O8), 3.16 Å,
Arg175 (N
1)-HPG (O9), 3.06 Å, Arg176
(N
)-HPG (O8), 2.51 Å, Thr198 (O
1)-HPG (O9), 2.82
Å. The loss of enzymatic activity for R175A or R176A indicates that the
interactions with HPG are essential in hydrolyzing HPG. The finding of higher
Km value and very low enzymatic activity for
R175K or R176K indeed suggests the crucial role of the guanidinyl group of
Arg175 or Arg176 in binding to the carboxyl group of
HPG. Structural comparison between R175A and wild-type enzymes shows little
deviation (0.212 Å) in the overall C
atoms. There is, however,
significant conformational alteration within the binding pocket
(Fig. 5A). The most
apparent difference is that the orientation of the side chain of
Asn173 essentially switches to a different direction in R175A
(Asn173 (N
)-Lys127 (N
), 2.97 Å in
R175A versus 6.40 Å in wild-type D-NCAase). Other
lesser variations such as the S
atom of Cys172, slightly
apart from that in the wild-type structure (0.61 Å), are also observed.
These results thus collectively suggest that Arg175 and
Arg176 are critical in maintaining a proper conformation to fit a
substrate with a carboxyl group, hence determining the substrate specificity.
We also examine the structure of N173A mutant that did not completely lose its
relative activity (5%). As shown in Table
III, N173A shares a homologous overall structure. Within the
binding pocket, N173A also shows minor conformational alteration
(Fig. 5B), indicating
that Asn173 is much less important in maintaining a conformation
for substrate binding, unlike that for R175A. In contrast, there is tighter
substrate binding affinity upon substitution of Asn173 with alanine
(
2.5-fold lower Km). One possible
interpretation is that the much larger side chain of Asn173 that
protrudes outward the pocket may hinder the docking of a substrate into the
right orientation toward the presumed reactive S
atom of its neighbor
Cys172. It is nevertheless noted that the S
atom of
Cys172 points away from the original position (0.37 Å) in
N173A, which may explain why it had lower relative activity and
kcat (8-fold reduced kcat).

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FIG. 5. Analysis of the active center for R175A and N173A. A,
superposition of active-site residues of wild-type (green) with those
of R175A (red). B, superposition of active-site residues of
wild-type (green) with those of N173A (red). Residues are
shown by stick structures. The oxygen, nitrogen, and sulfur atoms are red,
blue, and yellow, respectively.
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For the carbamoyl moiety, there are 14 interactions (Glu47,
Lys127, His144, Glu146, Ser172,
Asn173, and Asn197) including seven hydrogen bonds in
the C172S·HPG structure. Among these, the hydroxyl group of
Ser172 extending from the carboxyl end of a
-strand (residues
164 to 170) sits at the very bottom of this pocket and points directly to the
C7 atom (2.92 Å) of the carbamoyl moiety of HPG
(Fig. 4C). Side chains
of two nearby residues, Glu47 and Lys127, cluster around
that of Ser172 and face together as a triad
(Ser172-Glu47-Lys127) toward the carbamoyl
group; the side chain of Glu47 is situated close the N atom of the
carbamoyl moiety (Glu47 (O
1)-HPG (N12), 3.05 Å;
Glu47 (O
2)-HPG (N12), 3.33 Å), whereas the N
atom
of Lys127 sits near the O13 atom of the carbamoyl moiety (2.98
Å) (Fig. 4C).
Several interactions are observed among polar groups of Glu47,
Lys127, and Ser172 (Glu47
(O
1)-Ser172 (O
), 3.39 Å; Glu47
(O
2)-Ser172 (O
), 3.99 Å; and Lys127
(N
)-Ser172 (O
), 3.76 Å), which may facilitate to
polarize Ser172 O
atom in C172S or Cys172
S
atom of wild-type enzyme. Taken together, these results suggest that
the clustered Cys172-Glu47-Lys127 triad forms
a robust platform to catalyze an amidohydrolytic reaction when binding to a
substrate such as HPG; Cys172 with a nucleophilic S
atom
plays a key role in directly attacking the C7 atom of the carbamoyl group,
Glu47 acts as a general base, and Lys127 stabilizes a
tetrahedral transition state. A possible catalytic mechanism that consists of
two steps is thereby proposed: (i) an acylation reaction with the carbamoyl
moiety of substrate to cleave the susceptible C-N bond and the production of
an NH3 molecule, and (ii) deacylation of the acyl-enzyme
intermediate to yield a D-amino acid and a CO2 molecule
(10,
11).
The carboxyl group of Glu146 also interacts with the catalytic
triad via five interactions including a hydrogen bond with Lys127
(Glu146 (O
2)Lys127 (N
2), 2.65
Å). An imidazole ring from His144 sits just above the side
chain of Glu146 (His144 (N
2)-Glu146
(O
2), 2.58 Å), thus making a hydrogen network and fixing the
side-chain geometry of Lys127, His144, and
Glu146. The finding that H144A had a significant drop in the
relative activity (11) and
that Glu146 makes a hydrogen bond with the carbamoyl group of HPG
(Glu146 (O
2)HPG (N12), 2.97 Å in
C172S·HPG complex) suggest the role of His144 and
Glu146 in maintaining the binding pocket, as well as in supporting
the docking of a substrate.
Apart from those, Phe53, Pro131, Asn197,
Pro199, His201, and Asn202 located on three
loops (4661, 127146, 197206) are also in close proximity
to the substrate. The O atom at the main chain of Asn197 forms a
hydrogen bond (2.90 Å) with the N12 atom of the carbamoyl moiety of the
substrate, whereas Phe53 and Pro131 make four van der
Waals contacts (≤4.0 Å) with the carbamoyl group. Pro199,
His201, and Asn202 from a nearby loop (197206)
interacts with the hydroxyphenyl group of HPG that points to the outside space
of the binding pocket. It is noted that there is only one strong interaction
(Asn202 (N
2)-HPG (O15), 3.13 Å)
(Fig. 4C). In the
C172A-substrate structure, comparable interactions are also found. The large
volume enclosing the hydroxyphenyl group for more van der Waals contacts
suggests that D-NCAase favors a substrate with a long/bulky
hydrophobic side chain. Indeed, D-NCAase shows broad substrate
specificity toward N-carbamoyl-D-amino acid and hydrolyzes
better for larger substrates including D-phenylglycine,
D-methionine, and D-leucine
(6,
8). The finding that
D-NCAase had no detectable activity for a small substrate like
N-carbamoyl-glycine (data not shown) supports this model. In model
simulation analysis, an L-enantiomer also bumps onto the
127146 loop by fitting the carbamoyl group into the active site (data
not shown), consistent with its substrate requirement at the
D-enantiomeric form
(57).
Comparison of the Binding Pocket among Nitrilase, CSHase, and
D-NCAaseAlthough D-NCAase shares low
sequence homology with other D-NCAases from other species, the
Cys172-Glu47-Lys127 triad is all conserved
(11). Another member of the
nitrilase superfamily, the Nit domain of C. elegans NitFhit protein,
shows the same four-layer
-
-
-
fold with a
14.0-Å deviation in the overall C
atoms
(Fig. 6A, left
panel), despite lower sequence identity (25%)
(13). In support of their
related catalytic function, a common C-E-K catalytic triad is seen for
D-NCAase and Nit that both belong to the nitrilase superfamily
(14) with slight deviation in
the polar carboxyl group. It is nevertheless noted that the reactive thiol
group of the active cysteine points to different direction
(Fig. 6B). Further
differences in other regions of the binding pocket are observed. For instance,
side chains of Arg175 and Arg176 that are responsible
for interacting with the carboxyl moiety of a substrate in D-NCAase
are occupied with those of Val172 and Arg173 in Nit
(Fig. 6B), suggesting
that Nit would have its own substrate specificity. We have also compared the
D-NCAase structure with that of CSHase, the only other enzyme with
available structural coordinates that catalyzes the amidohydrolytic reaction
(11,
30). Even though CSHase has a
distinct structural architecture (three-layer
-
-
fold) and
presents the binding pocket in a different way
(Fig. 6A, right
panel), superposition of the catalytic triad between D-NCAase
and CSHase reveals a homologous catalytic triad
(Cys172-Glu47-Lys127 in D-NCAase
versus Cys177-Asp51-Lys144 in
CSHase) (Fig. 6C), in
accordance with the related hydrolytic reaction. However, other regions of the
binding pocket are essentially different; the residues proposed to bind to the
carboxyl moiety in CSHase are from a C-terminal fragment (Arg202)
and that of its neighbor subunit B (Lys217), respectively, as
compared with Arg175 and Arg176 from the same loop in
D-NCAase. Moreover, a hydrophobic region containing
Phe63, Trp111, Ile115, and Leu120
that may bind to the N-methyl group of the carbamoylsarcosine
molecule is only seen in CSHase (Fig.
6C).

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|
FIG. 6. Structural comparison of D-NCAase with Nit or CSHase.
A, comparison of D-NCAase structure with that of Nit
(left panel) or CSHase (right panel). The protein backbones
of D-NCAase are green, and those of Nit or CSHase are
red. Three catalytic residues (Glu47, Lys127,
and Cys172 in D-NCAase; Glu54,
Lys127, and Cys169 in Nit; Asp51,
Lys144, and Cys177 in CSHase) are shown by stick
structures. B, superposition of active-site residues of
D-NCAase (green) with those of Nit (red).
C, superposition of active-site residues of D-NCAase
(green) with those of CSHase (red). Residues essential in
the catalysis and substrate binding are shown by stick structures. The oxygen,
nitrogen, and sulfur atoms are red, blue, and yellow,
respectively.
|
|
In conclusion, we have determined crystal structures of mutant
D-NCAases (C172A, C172S, N173A, and R175A), as well as
substrate-bound complexes (C172A·HPG and C712S·HPG). All
structures present the same four-layer sandwich architecture as that of the
wild-type D-NCAase. The substrate-bound forms reveal that the
carbamoyl group of the substrate makes direct contact with a robust catalytic
triad (Cys172-Glu47-Lys127) located at the
interior of a solvent-accessible cleft for an amidohydrolytic reaction.
Arg175 and Arg176 that are situated nearby
Cys172 play crucial roles in binding to the carboxyl moiety of a
substrate, as well as maintaining a stable binding platform. The finding that
substitution of Arg175 or Arg176 with alanine abolished
its enzymatic activity further supports this model. For the peripheral portion
of the binding pocket, only a substrate that endows a side chain at the
D-enantiomeric form can loosely fit into it. A larger side chain
can thus make more van der Waals contacts for an enhanced binding. The
comparable geometry of C-(D/E)-K triad seen among D-NCAase,
NitFhit, and CSHase suggests a robust and conserved catalytic platform for a
related chemical reaction, perhaps being a result of convergent evolution; the
increased nucleophilicity of the S
atom from the nearby polar groups
can therefore attack the C7 atom of a susceptible C7N3 bond
efficiently. On the other hand, the unique specificity of a particular
biocatalyst is acquired from divergence of other regions within the binding
pocket as seen from these structures. The elucidation of the structural basis
of D-NCAase substrate specificity may thus facilitate the design of
mutant enzymes with altered specificity. The catalytic activity and stability
of D-NCAase may be also improved by a rational approach.
 |
FOOTNOTES
|
---|
* This work was supported by National Science Council Grants
NSC91-3112-B-007-011, NSC91-2313-B-007-002, and NSC90-2311-B-007-002 and by
Minister of Education Program for Promoting Academic Excellence of
Universities Grant 89-B-FA04-1-4 (Taiwan). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶ ||
¶ To whom correspondence may be addressed. Tel./Fax:
886-422856215; E-mail:
whhsu{at}dragon.nchu.edu.tw.||
To whom correspondence may be addressed. Tel.: 886-3-5742766; Fax:
886-3-5742766 or 886-3-5717237; E-mail:
wcwang{at}life.nthu.edu.tw.
1 The abbreviations used are: D-NCAase,
N-carbamoyl-D-amino acid amidohydrolase; HPG,
N-carbamoyl-D-p-hydroxyphenylglycine; CSHase,
N-carbamoylsarcosine amidohydrolase. 
 |
ACKNOWLEDGMENTS
|
---|
We acknowledge access to Macromolecular x-ray Crystallographic Center of
NTHU Instrument Center at Hsinchu, National Tsing Hua University, Taiwan for
data collection. We are grateful for the access to the following beamlines for
synchrotron data collection: BL-6A at the High Energy Accelerator Research
Organization (KEK), Photon Factory, Tsukuba, Japan, BL17B2 beamline at the
National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan, and
BL12B2 Taiwan beamline at Spring-8, Sayo, Japan.
 |
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