MANHY_PSEPU
ID MANHY_PSEPU Reviewed; 507 AA.
AC Q84DC4;
DT 11-JAN-2011, integrated into UniProtKB/Swiss-Prot.
DT 01-JUN-2003, sequence version 1.
DT 25-MAY-2022, entry version 50.
DE RecName: Full=Mandelamide hydrolase {ECO:0000303|PubMed:12670968, ECO:0000303|PubMed:15196015, ECO:0000303|PubMed:19074156, ECO:0000312|EMBL:AAO23019.1};
DE EC=3.5.1.86;
GN Name=mdlY {ECO:0000312|EMBL:AAO23019.1};
OS Pseudomonas putida (Arthrobacter siderocapsulatus).
OC Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales;
OC Pseudomonadaceae; Pseudomonas.
OX NCBI_TaxID=303;
RN [1] {ECO:0000305, ECO:0000312|EMBL:AAO23019.1}
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION, CATALYTIC ACTIVITY,
RP BIOPHYSICOCHEMICAL PROPERTIES, ACTIVITY REGULATION, SUBUNIT, INDUCTION, AND
RP MASS SPECTROMETRY.
RC STRAIN=ATCC 12633 / DSM 291 / JCM 13063 / CCUG 12690 / LMG 2257 / NBRC
RC 14164 / NCIMB 9494 / NCTC 10936 / VKM B-2187 / Stanier 90;
RX PubMed=12670968; DOI=10.1128/jb.185.8.2451-2456.2003;
RA McLeish M.J., Kneen M.M., Gopalakrishna K.N., Koo C.W., Babbitt P.C.,
RA Gerlt J.A., Kenyon G.L.;
RT "Identification and characterization of a mandelamide hydrolase and an
RT NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC
RT 12633.";
RL J. Bacteriol. 185:2451-2456(2003).
RN [2] {ECO:0000305}
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, MASS
RP SPECTROMETRY, AND MUTAGENESIS OF LYS-100; SER-180; SER-181 AND SER-204.
RX PubMed=15196015; DOI=10.1021/bi049907q;
RA Gopalakrishna K.N., Stewart B.H., Kneen M.M., Andricopulo A.D.,
RA Kenyon G.L., McLeish M.J.;
RT "Mandelamide hydrolase from Pseudomonas putida: characterization of a new
RT member of the amidase signature family.";
RL Biochemistry 43:7725-7735(2004).
RN [3] {ECO:0000305}
RP FUNCTION, CATALYTIC ACTIVITY, AND MUTAGENESIS OF THR-31; GLY-202; GLN-207;
RP SER-316; GLN-382 AND ILE-437.
RX PubMed=19074156; DOI=10.1093/protein/gzn073;
RA Wang P.F., Yep A., Kenyon G.L., McLeish M.J.;
RT "Using directed evolution to probe the substrate specificity of mandelamide
RT hydrolase.";
RL Protein Eng. Des. Sel. 22:103-110(2009).
CC -!- FUNCTION: Hydrolyzes both the R- and the S-enantiomers of mandelamide,
CC and phenylacetamide. Has lower activity on 3-phenylpropionaide and
CC lactamide. Does not hydrolyze benzamide. Hydrolyzes esters and amides
CC with little steric bulk. Preferentially hydrolyzes aromatic substrates.
CC {ECO:0000269|PubMed:12670968, ECO:0000269|PubMed:15196015,
CC ECO:0000269|PubMed:19074156}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(R)-mandelamide + H2O = (R)-mandelate + NH4(+);
CC Xref=Rhea:RHEA:22876, ChEBI:CHEBI:15377, ChEBI:CHEBI:17352,
CC ChEBI:CHEBI:28938, ChEBI:CHEBI:32382; EC=3.5.1.86;
CC Evidence={ECO:0000269|PubMed:12670968, ECO:0000269|PubMed:15196015,
CC ECO:0000269|PubMed:19074156};
CC -!- ACTIVITY REGULATION: Inhibited by 3,4-dichloroisocoumarin and PMSF.
CC {ECO:0000269|PubMed:12670968}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=34.2 uM for (R)-mandelamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:12670968};
CC KM=19.8 uM for (S)-mandelamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:12670968};
CC KM=33.1 uM for (R)-mandelamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC KM=19.9 uM for (S)-mandelamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC KM=3.8 uM for 2-phenylacetamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC KM=49 uM for 3-phenylpropionamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC KM=357 uM for (R)-2-methoxy-2-phenylacetamide (at 30 degrees Celsius,
CC pH 7.8) {ECO:0000269|PubMed:15196015};
CC KM=32 uM for N-methyl phenylacetamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC KM=170 uM for (R,S)-methyl mandelate (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC KM=288 uM for hexanoamide (at 30 degrees Celsius, pH 7.8)
CC {ECO:0000269|PubMed:15196015};
CC pH dependence:
CC Optimum pH is 7.8. {ECO:0000269|PubMed:12670968,
CC ECO:0000269|PubMed:15196015};
CC -!- SUBUNIT: Monomer. {ECO:0000269|PubMed:12670968}.
CC -!- INDUCTION: By growth on R,S-mandelamide, and to a lesser extent
CC mandelate. {ECO:0000269|PubMed:12670968}.
CC -!- MASS SPECTROMETRY: Mass=53820; Mass_error=4; Method=Electrospray;
CC Evidence={ECO:0000269|PubMed:12670968, ECO:0000269|PubMed:15196015};
CC -!- MASS SPECTROMETRY: Mass=53824; Method=Electrospray;
CC Evidence={ECO:0000269|PubMed:12670968, ECO:0000269|PubMed:15196015};
CC -!- SIMILARITY: Belongs to the amidase family. {ECO:0000255}.
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DR EMBL; AY143338; AAO23019.1; -; Genomic_DNA.
DR RefSeq; WP_016501744.1; NZ_UGUX01000003.1.
DR AlphaFoldDB; Q84DC4; -.
DR SMR; Q84DC4; -.
DR GeneID; 45526284; -.
DR BRENDA; 3.5.1.86; 5092.
DR GO; GO:0050537; F:mandelamide amidase activity; IDA:UniProtKB.
DR Gene3D; 3.90.1300.10; -; 1.
DR InterPro; IPR000120; Amidase.
DR InterPro; IPR020556; Amidase_CS.
DR InterPro; IPR023631; Amidase_dom.
DR InterPro; IPR036928; AS_sf.
DR PANTHER; PTHR11895; PTHR11895; 1.
DR Pfam; PF01425; Amidase; 1.
DR SUPFAM; SSF75304; SSF75304; 1.
DR PROSITE; PS00571; AMIDASES; 1.
PE 1: Evidence at protein level;
KW Hydrolase.
FT CHAIN 1..507
FT /note="Mandelamide hydrolase"
FT /id="PRO_0000403312"
FT ACT_SITE 100
FT /note="Charge relay system"
FT /evidence="ECO:0000250|UniProtKB:P97612"
FT ACT_SITE 180
FT /note="Charge relay system"
FT /evidence="ECO:0000250|UniProtKB:P97612"
FT ACT_SITE 204
FT /note="Acyl-ester intermediate"
FT /evidence="ECO:0000250|UniProtKB:P97612"
FT MUTAGEN 31
FT /note="T->I: More active on the (S)-enantiomers of
FT mandelamide and lactamide than the (R)-enantiomers; when
FT associated with N-437."
FT /evidence="ECO:0000269|PubMed:19074156"
FT MUTAGEN 100
FT /note="K->A: Abolishes activity on mandelamide."
FT /evidence="ECO:0000269|PubMed:15196015"
FT MUTAGEN 180
FT /note="S->A: Significantly decreases activity on
FT mandelamide."
FT /evidence="ECO:0000269|PubMed:15196015"
FT MUTAGEN 181
FT /note="S->A: Significantly decreases activity on
FT mandelamide."
FT /evidence="ECO:0000269|PubMed:15196015"
FT MUTAGEN 202
FT /note="G->A: Increase in KM values for aromatic substrates,
FT but not aliphatic substrates. Active against lactamide but
FT not against mandelamide; when associated with H-207 and E-
FT 382."
FT /evidence="ECO:0000269|PubMed:19074156"
FT MUTAGEN 202
FT /note="G->V: Increase in KM values for aromatic substrates,
FT but not aliphatic substrates."
FT /evidence="ECO:0000269|PubMed:19074156"
FT MUTAGEN 204
FT /note="S->A: Abolishes activity on mandelamide."
FT /evidence="ECO:0000269|PubMed:15196015"
FT MUTAGEN 207
FT /note="Q->H: Increases activity on lactamide, does not
FT affect activity on mandelamide; when associated with E-382.
FT Active against lactamide but not against mandelamide; when
FT associated with A-202 and E-382. More active on the (S)-
FT enantiomers of mandelamide and lactamide than the (R)-
FT enantiomers; when associated with S-316 and N-437."
FT /evidence="ECO:0000269|PubMed:19074156"
FT MUTAGEN 316
FT /note="S->N: More active on the (S)-enantiomers of
FT mandelamide and lactamide than the (R)-enantiomers; when
FT associated with H-207 and N-437."
FT /evidence="ECO:0000269|PubMed:19074156"
FT MUTAGEN 382
FT /note="Q->H: Increases activity on lactamide, does not
FT affect activity on mandelamide; when associated with H-207.
FT Active against lactamide but not against mandelamide; when
FT associated with A-202 and H-207."
FT /evidence="ECO:0000269|PubMed:19074156"
FT MUTAGEN 437
FT /note="I->N: More active on the (S)-enantiomers of
FT mandelamide and lactamide than the (R)-enantiomers. More
FT active on the (S)-enantiomers of mandelamide and lactamide
FT than the (R)-enantiomers; when associated with I-31. More
FT active on the (S)-enantiomers of mandelamide and lactamide
FT than the (R)-enantiomers; when associated with H-207 and N-
FT 316."
FT /evidence="ECO:0000269|PubMed:19074156"
SQ SEQUENCE 507 AA; 53814 MW; C1E7858848F76871 CRC64;
MRHPVDMPEK VGTDAKRLFA QPEHLWELTL TEASALVRHR RITSRQLVEA WLSRIADFSE
LNAFISVDAA AALKQADSYD HYLEAGGDPL PLGGVPIAVK DNIQVVGFAN TAGTPALSKF
FPTCNARVIE PLLKAGAIVV GKTNMHELAF GTSGYNTAYH IPGVIGVRNA FDHSCIAGGS
SSGSGTAVGA LLIPAALGTD TGGSVRQPGA VNGCVGFRPT VGRYPVDGIT PISPTRDTPG
PIARSVEDIV LLDSIITGAL PAEVPAAESI RLGVVDQLWA DLSEPVRKLT EDALRKLEQQ
GVQIVRVSMS EIFEMSHAVS MPLALHECRS ALTEYLSANE TGVSFDELVA GISSPDVRTI
FEDYILPGRL GELEGQSVDL EQAYATAMKD ARPKLIQSFE FLFKEHQLDA IIHPTTPDLA
IKSNPAATSF EAFARMIRNA DPASNAGMPG ISLPAGLSQQ EGLPVGIEIE GLPGSDARLL
SIANFIESIL GRGPTPTRSG VESKISM
