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• W2002604337 abstract "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 α-β-β-α 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 k cat or K m, whereas R175K or R176K showed high K m and very low k cat 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. 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 α-β-β-α 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 k cat or K m, whereas R175K or R176K showed high K m and very low k cat 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. The enzyme N-carbamoyl-d-amino acid amidohydrolase (d-NCAase) 1The abbreviations used are: d-NCAase, N-carbamoyl-d-amino acid amidohydrolase; HPG, N-carbamoyl-d-p-hydroxyphenylglycine; CSHase, N-carbamoylsarcosine amidohydrolase.1The abbreviations used are: d-NCAase, N-carbamoyl-d-amino acid amidohydrolase; HPG, N-carbamoyl-d-p-hydroxyphenylglycine; CSHase, N-carbamoylsarcosine amidohydrolase. hydrolyzes N-carbamoyl-d-amino acids to d-amino acids, carbon dioxide, and ammonia (1Syldatk C. Läufer A. Müller R. Höke H. Adv. Biochem. Eng. Biotechnol. 1990; 41: 30-75Google Scholar). Several microorganisms produce d-NCAase activity including Agrobacterium (2Olivieri R. Fascetti E. Angelini L. Degen L. Biotechnol. Bioeng. 1981; 23: 2173-2183Crossref Scopus (152) Google Scholar, 3Runser S. Chinski N. Ohleyer E. Appl. Microbiol. Biotechnol. 1990; 33: 382-388Crossref Scopus (86) Google Scholar, 4Nanba H. Ikenaka Y. Yamada Y. Yajima K. Takano M. Takahashi S. Biosci. Biotechnol. Biochem. 1998; 62: 875-881Crossref PubMed Scopus (63) Google Scholar), Arthrobacter (5Moller A. Syldatk C. Schulze M. Wagner F. Enzyme Microb. Technol. 1988; 10: 618-625Crossref Scopus (109) Google Scholar), Comamonas (6Ogawa J. Shimizu S. Yamada H. Eur. J. Biochem. 1993; 212: 685-691Crossref PubMed Scopus (64) Google Scholar), and thermotolent bacteria such as Blastobacter sp. A17p-4 (7Ogawa J. Chung M.C.-M. Hida S. Yamada H. Shimizu S. J. Biotechnol. 1994; 38: 11-19Crossref PubMed Scopus (77) Google Scholar) and Pseudomonas sp. strain KNK003A (8Ikenaka Y. Nanba H. Yamada Y. Yajima K. Takano M. Takahashi S. Biosci. Biotechnol. Biochem. 1998; 62: 882-886Crossref PubMed Scopus (45) Google Scholar). Despite low sequence identities among different species, d-NCAases require a strict d-enantiomer of the N-carbamoyl-amino acid as their substrate (5Moller A. Syldatk C. Schulze M. Wagner F. Enzyme Microb. Technol. 1988; 10: 618-625Crossref Scopus (109) Google Scholar, 6Ogawa J. Shimizu S. Yamada H. Eur. J. Biochem. 1993; 212: 685-691Crossref PubMed Scopus (64) Google Scholar, 7Ogawa J. Chung M.C.-M. Hida S. Yamada H. Shimizu S. J. Biotechnol. 1994; 38: 11-19Crossref PubMed Scopus (77) Google Scholar). 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 β-lactam antibiotics by a second enzymatic step catalyzed by d-NCAase (2Olivieri R. Fascetti E. Angelini L. Degen L. Biotechnol. Bioeng. 1981; 23: 2173-2183Crossref Scopus (152) Google Scholar, 9Takahashi S. Ohashi T. Kii Y. Kumagai H. Yamada H. J. Ferment. Technol. 1979; 57: 328-332Google Scholar).Crystal structure of d-NCAase reveals a tetramer with 222 symmetry; each monomer shows a four-layer α-β-β-α sandwich fold (10Nakai T. Hasegawa T. Yamashita E. Yamamoto M. Kumasaka T. Ueki T. Nanba H. Ikinaka Y. Takahashi S. Sato M. Tsukihara T. Structure. 2000; 8: 729-739Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (70) Google Scholar). 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 (12Grifantini R. Pratesi C. Galli G. Grandi G. J. Biol. Chem. 1996; 271: 9326-9331Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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) (11Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (70) Google Scholar). Interestingly, the Nit domain of Caenorhabditis elegans NitFhit protein (13Pace H.C. Hodawadekar S.C. Draganescu A. Huang J. Bieganowski P. Pekarsky Y. Croce C.M. Brenner C. Curr. Biol. 2000; 10: 907-917Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) 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 (14Pace H.C. Brenner C. Genome Biology. 2001; (http://genomebiology.com/2001/2/1/reviews/0001)PubMed Google Scholar). 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 (10Nakai T. Hasegawa T. Yamashita E. Yamamoto M. Kumasaka T. Ueki T. Nanba H. Ikinaka Y. Takahashi S. Sato M. Tsukihara T. Structure. 2000; 8: 729-739Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (70) Google Scholar). 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 PROCEDURESSite-directed Mutagenesis—Site-directed mutagenesis was carried out using a Transformer™ 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 (15Hsu W.H. Chien F.T. Hsu C.L. Wang T.C. Yuan H.S. Wang W.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 694-695Crossref PubMed Scopus (8) Google Scholar). The purified protein was analyzed by a SDS-PAGE gel to verify the purity. The protein concentration was assayed according to the Bradford method (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) 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) (17Gordon S.A. Fleck A. Bell J. Ann. Clin. Biochem. 1978; 15: 270-275Crossref PubMed Scopus (31) Google Scholar). The K m (mm) and k cat (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 K m).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 (T m) curve was normalized according to the highest CD signal as 1 and the lowest CD signal as 0. T m 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 (15Hsu W.H. Chien F.T. Hsu C.L. Wang T.C. Yuan H.S. Wang W.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 694-695Crossref PubMed Scopus (8) Google Scholar). 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 × 0.4 × 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 (11Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (70) Google Scholar).Table IData collection and refinement statisticsData collection statisticsData setC172AC172SC172A·HPGC172S·HPGR175AN173ASourceCuKαKEK BL-6AaBeamline (BL) 6A at High Energy Accelerator Research Organization. KEK Photon Factory, Tsukuba, JapanKEK BL-6AKEK BL-6ASPring-8 BL-12BbBL (BL) 12B2 Taiwan beamline at Japan Synchrotron Radiation Research Institute (JASRI), Spring-8, Sayo, JapanSPring-8 BL-12BCell dimensionsa = 69.50 Åa = 72.98 Åa = 69.24 Åa = 69.74 Åa = 71.00 Åa = 68.65 Åb = 67.77 Åb = 67.45 Åb = 68.10 Åb = 67.97 Åb = 67.77 Åb = 67.69 Åc = 138.26 Åc = 137.68 Åc = 138.34 Åc = 138.36 Åc = 136.95 Åc = 138.50 Åβ = 96.08°β = 98.86°β = 96.27°β = 96.12°β = 95.93°β = 95.96°Resolution (Å)2.002.202.002.402.001.95Highest resolution shell (Å)2.07-2.002.32-2.202.11-2.002.53-2.402.07-2.002.02-1.95Completeness (%)cValues in parentheses refer to statistics in the highest resolution shell98.9 (98.9)99.8 (99.8)95.2 (95.2)96.0 (96.0)90.7 (98.7)99.8 (99.9)Average I/σ(I)5.65.27.05.17.17.1R merge (%)dR merge = Σ|Ia-〈I〉|/ΣIn7.912.58.013.29.210.3Unique reflections81,35563,92178,31145,95979,50292,630Refinement statisticsResolution (Å)30.0-2.030.0-2.230.0-2.030.0-2.425.0-2.025.0-1.95Protein atoms9,5609,5649,6209,6249,5409,548Solvent atoms1,1558187695287141,154Substrate atoms6060R eR = Σ|Fo-Fo|/ΣFo , where Fo and Fc are the observed and calculated structure-factor amplitudes, respectively0.1790.1880.1750.1860.1900.155R freefR free was computed using 5% of the data assigned randomly0.2350.2670.2330.2650.2460.209r.m.s.d. bond length (Å)gr.m.s.d., root mean square deviation0.0370.0200.0230.0320.0310.028r.m.s.d. bond angles (°)2.661.771.962.422.252.12r.m.s.d. torsion angles (°)8.077.567.678.547.777.30Estimated coordinate error (Å)0.1940.2880.1930.6360.2230.151a Beamline (BL) 6A at High Energy Accelerator Research Organization. KEK Photon Factory, Tsukuba, Japanb BL (BL) 12B2 Taiwan beamline at Japan Synchrotron Radiation Research Institute (JASRI), Spring-8, Sayo, Japanc Values in parentheses refer to statistics in the highest resolution shelld R merge = Σ|Ia-〈I〉|/ΣIne R = Σ|Fo-Fo|/ΣFo , where Fo and Fc are the observed and calculated structure-factor amplitudes, respectivelyf R free was computed using 5% of the data assigned randomlyg r.m.s.d., root mean square deviation Open table in a new tab 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 CuKα 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 (18Leslie A.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1696-1702Crossref PubMed Scopus (485) Google Scholar) and the CCP4 program suites (19CollaborativeActa Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar). 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 (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar). 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) was used to calculate a difference Fourier map with the coefficients 2F o - F c and calculated phases for each mutant or mutant-substrate complex. A tetramer with the α/β 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 (21Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar).Structure refinement was carried out with the REFMAC5 program (22Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar). 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 (R free) for cross-validation of the model. Sigma A-weighted 2F o - F c and F o - F c 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 R free. 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 (23Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2562) Google Scholar), water molecules were introduced automatically into the model. TLS refinement (24Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1646) Google Scholar) prior to individual isotropic B value refinement was used to further reduce the R and R free values. The stereochemistry of the protein model was assessed using the program PROCHECK (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Estimates of the coordinate errors were made using the method of Read (26Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2035) Google Scholar). 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 (27Kleywegt G.J. Jones T.A. Methods Enzymol. 1997; 277: 525-545Crossref PubMed Scopus (303) Google Scholar) by superimposing overall Cα atoms of a monomer. For binding site comparison, Cα 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) or CSHase (Protein Data Bank code 1NBA) 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 (28Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) coupled to RASTER3D (29Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar) programs. The figures of electron density map were prepared with PyMOL (www.pymol.org).RESULTS AND DISCUSSIONExpression and Enzymatic Analysis of d-NCAase Mutants— Based on the d-NCAase·HPG model (11Wang W.C. Hsu W.H. Chien F.T. Chen C.Y. J. Mol. Biol. 2001; 306: 251-261Crossref PubMed Scopus (70) Google Scholar), 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.N173A, R175K, and R176K were subjected for kinetic analysis. As shown in Table II, R175K and R176K had ∼2.5- and 4-fold higher K m value, respectively, as compared with that of wild-type (Table II). Moreover, the k cat value was significantly reduced for either of two, resulting in an extremely lower k cat/K m value than that of wild-type. The N173A mutant had ∼13-fold reduced k cat but 2.5-fold lower K m.Table IIKinetic parameters and T m values for wild-type and mutant D-NCAasesd-NCAasekcatK mk cat/K mT mmin -1mMmin -1 mM -1°CWild-type5.2×1021.34.0×10263C172ANo activityC172SNot determined, less than 0.1% activity70N173A3.8×1010.527.3×10168R175ANo activityR176ANo activity71R175K2.7×10-13.28.5×10-265R176K6.35.41.165 Open table in a new tab 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 T m 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% (R free = 26.7%) (Table I). Similarly, the structure of C172A was determined and refined to 2.0 Å resolution, with an R of 17.9% (R free = 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%, R free = 24.6%) and 1.95 Å (R = 15.5%, R free = 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 α-β-β-α architecture with modest deviation in the overall Cα atoms (Table III).Fig. 2The 2F o - F c electron density map of d-NCAase mutant around residue 172. A, C172S mutant. B, C172A mutant. Maps are contoured at the 1.5-σ level.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIComparison of the d-NCAase monomer and binding-site region Comparison of root mean square deviations (Å) for the overall Cα atoms in monomer A, and the Cα 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.Root mean square deviation (Å)d-NCAaseCα atoms in monomer AaHomologous Cα atoms are compared between superimposed structuresCα atoms in binding-site regionbThe residues in binding site for analysis are Glu47, Lys127, His144, Glu146, Cys172, Asn173, Arg175, Arg176, Asn197, Thr198, His201, and Asn202All atoms in binding-site regionbThe residues in binding site for analysis are Glu47, Lys127, His144, Glu146, Cys172, Asn173, Arg175, Arg176, Asn197, Thr198, His201, and Asn202WTcWT, wild-type enzyme vs. C172A0.2620.1500.530WT vs. C172A·HPG0.3220.2020.493C172A vs. C172A·HPG0.2280.1540.359WT vs. C172S0.2020.1470.252WT vs. C172S·HPG0.2990.1940.662C172S vs. C172S·HPG0.3270.2260.647WT vs. N173A0.2770.0950.472WT vs. R175A0.2120.2371.079a Homologous Cα atoms are compared between superimposed structuresb The residues in binding site for analysis are Glu47, Lys127, His144, Glu146, Cys172, Asn173, Arg175, Arg176, Asn197, Thr198, His201, and Asn202c WT, wild-type enzyme Open table in a new tab The C172A·HPG and C172S·HPG structures were determined and refined to an R of 17.5% (R free = 23.3%) and 18.6% (R free = 26.5%), respectively (Table I). As seen in Fig. 3A, the 2F o - F c 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 α/β-type structure with two central β 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 Cα 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).Fig. 3Crystal structure of the C172S-substrate complex. A, the 2F o - F c map of the C172S·HPG complex around HPG, contoured at the 1.5-σ 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 Cα 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 (Oγ)-sulfate" @default.
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• W2002604337 title "Structural Basis for Catalysis and Substrate Specificity of Agrobacterium radiobacter N-Carbamoyl-D-amino Acid Amidohydrolase" @default.
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