Structural Basis for Catalysis and Substrate Specificity of Agrobacterium radiobacter N-Carbamoyl-D-amino Acid Amidohydrolase*

Cheng-Yu Chen {ddagger}, Wei-Chun Chiu {ddagger}, Jai-Shin Liu {ddagger}, Wen-Hwei Hsu  § ¶ || and Wen-Ching Wang  {ddagger} ¶ ||

From the {ddagger}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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 {alpha}-{beta}-{beta}-{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 {beta}-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 {alpha}-{beta}-{beta}-{alpha} 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—Site-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 Enzymes—The 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 Assays—The 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 (1–10-fold Km).

Circular Dichroism of Wild-type and Mutant D-NCAases—CD 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.

Crystallization—D-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).


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TABLE I
Data collection and refinement statistics

 

Data Collection and Processing—For 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{alpha} 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 Refinement—The 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 {alpha}/{beta} 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{alpha} atoms of a monomer. For binding site comparison, C{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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.

 

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.


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TABLE II
Kinetic parameters and Tm values for wild-type and mutant D-NCAases

 

CD Spectroscopy of Wild-type and Mutant D-NCAases—CD 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·HPG—The crystal structure of C172S was determined to 2.2 Å by molecular replacement method. Residues 3–304 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 {alpha}-{beta}-{beta}-{alpha} architecture with modest deviation in the overall C{alpha} 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-{sigma} 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{alpha} atoms in monomer A, and the C{alpha} 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.

 

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 {alpha}/{beta}-type structure with two central {beta} 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{alpha} 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-{sigma} 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.

 

The Binding Pocket—The substrate is bound in a pocket surrounded by three large loops (46–61, 127–146, and 197–206) and a short loop (172–178). 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{alpha} 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{gamma})-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{gamma})-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.

 

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{gamma})-HPG (O9), 2.86 Å, Asn173 (N{delta}2)-HPG (O8), 3.16 Å, Arg175 (N{eta}1)-HPG (O9), 3.06 Å, Arg176 (N{epsilon})-HPG (O8), 2.51 Å, Thr198 (O{gamma}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{alpha} 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{delta})-Lys127 (N{zeta}), 2.97 Å in R175A versus 6.40 Å in wild-type D-NCAase). Other lesser variations such as the S{gamma} 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{gamma} atom of its neighbor Cys172. It is nevertheless noted that the S{gamma} 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.

 

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 {beta}-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{epsilon}1)-HPG (N12), 3.05 Å; Glu47 (O{epsilon}2)-HPG (N12), 3.33 Å), whereas the N{zeta} 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{epsilon}1)-Ser172 (O{gamma}), 3.39 Å; Glu47 (O{epsilon}2)-Ser172 (O{gamma}), 3.99 Å; and Lys127 (N{zeta})-Ser172 (O{gamma}), 3.76 Å), which may facilitate to polarize Ser172 O{gamma} atom in C172S or Cys172 S{gamma} 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{gamma} 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{epsilon}2)–Lys127 (N{zeta}2), 2.65 Å). An imidazole ring from His144 sits just above the side chain of Glu146 (His144 (N{epsilon}2)-Glu146 (O{epsilon}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{epsilon}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 (46–61, 127–146, 197–206) 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 (197–206) 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{delta}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 127–146 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-NCAase—Although 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 {alpha}-{beta}-{beta}-{alpha} fold with a 14.0-Å deviation in the overall C{alpha} 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 {alpha}-{beta}-{alpha} 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{gamma} atom from the nearby polar groups can therefore attack the C7 atom of a susceptible C7–N3 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. Back

¶ || 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. Back


    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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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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