true2022-02-232023-11-08108NSAR_AMYSPCloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.Tokuyama S.Hatano K.doi:10.1007/s0025300503471995Appl. Microbiol. Biotechnol.42884-889NUCLEOTIDE SEQUENCE [GENOMIC DNA]TS-1-60Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60.Tokuyama S.Hatano K.doi:10.1007/bf001911811995Appl. Microbiol. Biotechnol.42853-859PROTEIN SEQUENCE OF 1-24FUNCTION AS A RACEMASECATALYTIC ACTIVITYCOFACTORACTIVITY REGULATIONBIOPHYSICOCHEMICAL PROPERTIESSUBUNITUnexpected divergence of enzyme function and sequence: 'N-acylamino acid racemase' is o-succinylbenzoate synthase.Palmer D.R.Garrett J.B.Sharma V.Meganathan R.Babbitt P.C.Gerlt J.A.doi:10.1021/bi990140p1999Biochemistry384252-4258FUNCTIONCATALYTIC ACTIVITYACTIVITY REGULATIONBIOPHYSICOCHEMICAL PROPERTIESMUTAGENESIS OF LYS-163 AND LYS-263Evolution of enzymatic activity in the enolase superfamily: functional studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.Taylor Ringia E.A.Garrett J.B.Thoden J.B.Holden H.M.Rayment I.Gerlt J.A.doi:10.1021/bi035815+2004Biochemistry43224-229FUNCTIONCATALYTIC ACTIVITYBIOPHYSICOCHEMICAL PROPERTIESACTIVE SITESMUTAGENESIS OF LYS-163 AND LYS-263Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily.Glasner M.E.Fayazmanesh N.Chiang R.A.Sakai A.Jacobson M.P.Gerlt J.A.Babbitt P.C.doi:10.1016/j.jmb.2006.04.0552006J. Mol. Biol.360228-250FUNCTIONEVOLUTION OF THE O-SUCCINYLBENZOATE SYNTHASE/N-ACYLAMINO ACID RACEMASE FAMILYRole of an active site loop in the promiscuous activities of Amycolatopsis sp. T-1-60 NSAR/OSBS.McMillan A.W.Lopez M.S.Zhu M.Morse B.C.Yeo I.C.Amos J.Hull K.Romo D.Glasner M.E.doi:10.1021/bi500573v2014Biochemistry534434-4444FUNCTIONCATALYTIC ACTIVITYBIOPHYSICOCHEMICAL PROPERTIESMUTAGENESIS OF PRO-18; PHE-19; ARG-20; THR-21; SER-22; PHE-23; GLY-24 AND ASP-140Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis.Thoden J.B.Taylor Ringia E.A.Garrett J.B.Gerlt J.A.Holden H.M.Rayment I.doi:10.1021/bi04978972004Biochemistry435716-5727X-RAY CRYSTALLOGRAPHY (1.87 ANGSTROMS) IN COMPLEXES WITH 2-SUCCINYLBENZOIC ACID; N-ACETYL-METHIONINE; N-SUCCINYL-METHIONINE; N-SUCCINYL-PHENYLGLYCINE AND MAGNESIUMCOFACTORSUBUNITDOMAINACTIVE SITESAn improved racemase/acylase biotransformation for the preparation of enantiomerically pure amino acids.Baxter S.Royer S.Grogan G.Brown F.Holt-Tiffin K.E.Taylor I.N.Fotheringham I.G.Campopiano D.J.doi:10.1021/ja305438y2012J. Am. Chem. Soc.13419310-19313X-RAY CRYSTALLOGRAPHY (2.71 ANGSTROMS) OF MUTANT ASP-291/TYR-323 IN COMPLEX WITH N-ACETYL-METHIONINE AND MAGNESIUMFUNCTION AS A RACEMASECATALYTIC ACTIVITYBIOPHYSICOCHEMICAL PROPERTIESBIOTECHNOLOGYMUTAGENESIS OF GLY-291 AND PHE-323Structure of N-Acylamino Acid Racemase Mutants in Complex with Substrates.Sanchez-Carron G.Campopiano D.Grogan G.2015-10PDBX-RAY CRYSTALLOGRAPHY (2.08 ANGSTROMS) OF MUTANTS IN COMPLEXES WITH N-ACETYL NAPHTHYLALANINE; N-ACETYL PHENYLALANINE AND MAGNESIUM2.30A/B/C/D=1-3682.20A/B/C/D=1-3682.10A/B/C/D=1-3681.87A/B/C/D=1-3682.71A/B/C/D=1-3682.08A/B=1-3682.58A/B/C/D=1-3682.44A/B/C/D=1-3682.11A/B/C/D=1-3682.52A/B/C/D=1-3682.90A/B/C/D=1-368N-AcetylmethionineN-Succinyl MethionineN-Succinyl PhenylglycineO-Succinylbenzoate316NAAAREnolase-like C-terminal domainEnolase-like, N-terminal domainEnolase-like_C_sfEnolase-like_NEnolase_C-likeMandelate_racemase_CMandelate_racemase_N_domOSBS/NAAARmenC_lowGC_archO-SUCCINYLBENZOATE SYNTHASEO-SUCCINYLBENZOATE SYNTHASE-RELATEDMR_MLE_CMR_MLE_Nmuconate_cycloisomeraseN-succinylamino_acid_racemaseo-succinylbenzoate_synthaseMR_MLEEnolase C-terminal domain-likeEnolase N-terminal domain-likeAA sequenceN-succinylamino acid racemaseNSAR5.1.1.-N-acylamino acid racemaseAARNAAAR5.1.1.-o-succinylbenzoate synthaseOSB synthaseOSBS4.2.1.113AaarActs as a N-succinylamino acid racemase (NSAR) that catalyzes the racemization of N-succinyl-phenylglycine and N-succinyl-methionine (PubMed:14705949, PubMed:24955846). Can catalyze the racemization of a broad range of N-acylamino acids, including N-acetyl-D/L-methionine, N-propionyl-D/L-methionine, N-butyryl-D/L-methionine and N-chloroacetyl-L-valine (PubMed:7766084, PubMed:10194342, PubMed:14705949, PubMed:23130969). Also converts 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) to 2-succinylbenzoate (OSB) (PubMed:10194342, PubMed:14705949, PubMed:24955846). Catalyzes both N-succinylamino acid racemization and OSB synthesis at equivalent rates (PubMed:14705949, PubMed:24955846). NSAR is probably the biological function of this enzyme (Probable).Binds 1 divalent metal cation per subunit (PubMed:15134446). Enhanced by the addition of divalent metal ions such as Co(2+), Mn(2+) and Fe(2+) (PubMed:7766084).Inhibited by EDTA and sulfhydryl reagents such as p-chloromercuribenzoic acid (PubMed:7766084). Both OSBS and NAAAR activities are inhibited competitively by salicylhydroxamate (PubMed:10194342).kcat is 110 sec(-1) with N-succinyl-R/S-methionine as substrate (PubMed:14705949). kcat is 190 sec(-1) with N-succinyl-R/S-phenylglycine as substrate (PubMed:14705949). kcat is 42 sec(-1) with N-succinyl-L-phenylglycine as substrate (PubMed:24955846). kcat is 20 sec(-1) with N-acetyl-L-methionine as substrate (PubMed:23130969). kcat is 14 sec(-1) with N-acetyl-D-methionine as substrate (PubMed:23130969). kcat is 4.8 sec(-1) with N-acetyl-R-methionine as substrate (PubMed:14705949). kcat is 6.4 sec(-1) with N-acetyl-S-methionine as substrate (PubMed:14705949). kcat is 11.7 sec(-1) with N-acetyl-S-methionine as substrate (PubMed:10194342). kcat is 41 sec(-1) with N-acetyl-D-(4-fluoro)phenylglycine as substrate (PubMed:23130969). kcat is 97 sec(-1) with N-acetyl-allylglycine as substrate (PubMed:23130969). kcat is 120 sec(-1) with SHCHC as substrate (PubMed:10194342, PubMed:14705949). kcat is 46 sec(-1) with SHCHC as substrate (PubMed:24955846).6.5 mM for N-succinyl-R-methionine5.9 mM for N-succinyl-S-methionine0.94 mM for N-succinyl-R-phenylglycine0.95 mM for N-succinyl-S-phenylglycine1 mM for N-succinyl-L-phenylglycine18.5 mM for N-acetyl-L-methionine18 mM for N-acetyl-L-methionine11.3 mM for N-acetyl-D-methionine40 mM for N-acetyl-D-methionine9.8 mM for N-acetyl-R-methionine11 mM for N-acetyl-S-methionine35 mM for N-acetyl-D-(4-fluoro)phenylglycine0.48 mM for SHCHC0.55 mM for SHCHCOptimum pH is 7.5.Optimum temperature is 50 degrees Celsius. Stable at 55 degrees Celsius for 30 min.Homooctamer.The active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the racemase activity. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from E.coli lacks this functionality.Variants with increased racemase activity could be used on an industrial scale for the production of enantiomerically pure alpha-amino acids.Belongs to the mandelate racemase/muconate lactonizing enzyme family. MenC type 2 subfamily.N-succinylamino acid racemase394061368Proton donor163Proton acceptor263135161189191214239293Small increase in OSBS catalytic efficiency and 2-fold decrease in NSAR catalytic efficiency.A18200-fold decrease in OSBS catalytic efficiency and 120-fold decrease in NSAR catalytic efficiency. 2100-fold decrease in OSBS catalytic efficiency and 180-fold decrease in NSAR catalytic efficiency; when associated with A-23.A1932-fold decrease in OSBS catalytic efficiency and 8-fold decrease in NSAR catalytic efficiency.E204-fold decrease in OSBS and NSAR catalytic efficiencies.A213-fold increase in OSBS catalytic efficiency and 4-fold decrease in NSAR catalytic efficiency.R222100-fold decrease in OSBS catalytic efficiency and 180-fold decrease in NSAR catalytic efficiency; when associated with A-19.A23Small decrease in OSBS catalytic efficiency and 4-fold decrease in NSAR catalytic efficiency.A243-fold decrease in OSBS and NSAR catalytic efficiencies.R140Significantly impairs both NAAAR and OSBS activities.ARSSignificantly impairs both NAAAR and OSBS activities.RSShows up to 6-fold higher activity than the wild-type on a range of N-acetylated amino acids; when associated with Y-323.D291Shows up to 6-fold higher activity than the wild-type on a range of N-acetylated amino acids; when associated with D-291.Y323P537424650576069717778808390971141191231281361421551581701801841961981992032042082112222292362452532582622642682702822872892963063203223243263283303353403443463533593603661996-11-0113940628984d2d3ecee48a82a93e006313c007MKLSGVELRRVQMPLVAPFRTSFGTQSVRELLLLRAVTPAGEGWGECVTMAGPLYSSEYNDGAEHVLRHYLIPALLAAEDITAAKVTPLLAKFKGHRMAKGALEMAVLDAELRAHERSFAAELGSVRDSVPCGVSVGIMDTIPQLLDVVGGYLDEGYVRIKLKIEPGWDVEPVRAVRERFGDDVLLQVDANTAYTLGDAPQLARLDPFGLLLIEQPLEEEDVLGHAELARRIQTPICLDESIVSARAAADAIKLGAVQIVNIKPGRVGGYLEARRVHDVCAAHGIPVWCGGMIETGLGRAANVALASLPNFTLPGDTSASDRFYKTDITEPFVLSGGHLPVPTGPGLGVAPIPELLDEVTTAKVWIGStruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetruetrue