CARB_PECCC
ID CARB_PECCC Reviewed; 250 AA.
AC Q9XB60;
DT 26-NOV-2014, integrated into UniProtKB/Swiss-Prot.
DT 01-NOV-1999, sequence version 1.
DT 03-AUG-2022, entry version 65.
DE RecName: Full=Carboxymethylproline synthase {ECO:0000303|PubMed:15595850};
DE EC=2.3.1.226 {ECO:0000269|PubMed:14625287, ECO:0000269|PubMed:15595850};
DE AltName: Full=Carbapenem biosynthesis protein B {ECO:0000305};
GN Name=carB {ECO:0000303|PubMed:15595850};
OS Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora subsp.
OS carotovora).
OC Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales;
OC Pectobacteriaceae; Pectobacterium.
OX NCBI_TaxID=555;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RC STRAIN=ATCC 39048 / GS101 / SC 12 {ECO:0000312|EMBL:AAD38230.1};
RX PubMed=9402024; DOI=10.1046/j.1365-2958.1997.6001974.x;
RA McGowan S.J., Sebaihia M., O'Leary S., Hardie K.R., Williams P.,
RA Stewart G.S., Bycroft B.W., Salmond G.P.;
RT "Analysis of the carbapenem gene cluster of Erwinia carotovora: definition
RT of the antibiotic biosynthetic genes and evidence for a novel beta-lactam
RT resistance mechanism.";
RL Mol. Microbiol. 26:545-556(1997).
RN [2]
RP FUNCTION, CATALYTIC ACTIVITY, AND BIOPHYSICOCHEMICAL PROPERTIES.
RC STRAIN=ATCC 39048 / GS101 / SC 12;
RX PubMed=15595850; DOI=10.1021/bi0483662;
RA Gerratana B., Arnett S.O., Stapon A., Townsend C.A.;
RT "Carboxymethylproline synthase from Pectobacterium carotorova: a
RT multifaceted member of the crotonase superfamily.";
RL Biochemistry 43:15936-15945(2004).
RN [3]
RP FUNCTION.
RC STRAIN=ATCC 39048 / GS101 / SC 12;
RX PubMed=15726176; DOI=10.1039/b416423g;
RA Sorensen J.L., Sleeman M.C., Schofield C.J.;
RT "Synthesis of deuterium labelled L- and D-glutamate semialdehydes and their
RT evaluation as substrates for carboxymethylproline synthase (CarB)--
RT implications for carbapenem biosynthesis.";
RL Chem. Commun. (Camb.) 9:1155-1157(2005).
RN [4]
RP FUNCTION, CATALYTIC ACTIVITY, AND PATHWAY.
RX PubMed=14625287; DOI=10.1074/jbc.m311824200;
RA Sleeman M.C., Schofield C.J.;
RT "Carboxymethylproline synthase (CarB), an unusual carbon-carbon bond-
RT forming enzyme of the crotonase superfamily involved in carbapenem
RT biosynthesis.";
RL J. Biol. Chem. 279:6730-6736(2004).
RN [5]
RP FUNCTION, REACTION MECHANISM, AND MUTAGENESIS OF GLU-131.
RX PubMed=18972478; DOI=10.1002/anie.200803906;
RA Batchelar E.T., Hamed R.B., Ducho C., Claridge T.D., Edelmann M.J.,
RA Kessler B., Schofield C.J.;
RT "Thioester hydrolysis and C-C bond formation by carboxymethylproline
RT synthase from the crotonase superfamily.";
RL Angew. Chem. Int. Ed. Engl. 47:9322-9325(2008).
RN [6]
RP FUNCTION, AND MUTAGENESIS OF TRP-79; MET-108; GLN-111 AND HIS-229.
RX PubMed=21505494; DOI=10.1038/nchem.1011;
RA Hamed R.B., Gomez-Castellanos J.R., Thalhammer A., Harding D., Ducho C.,
RA Claridge T.D., Schofield C.J.;
RT "Stereoselective C-C bond formation catalysed by engineered
RT carboxymethylproline synthases.";
RL Nat. Chem. 3:365-371(2011).
RN [7]
RP X-RAY CRYSTALLOGRAPHY (2.24 ANGSTROMS) IN COMPLEX WITH ACETYL-COA, AND
RP SUBUNIT.
RX PubMed=16096274; DOI=10.1074/jbc.m507196200;
RA Sleeman M.C., Sorensen J.L., Batchelar E.T., McDonough M.A.,
RA Schofield C.J.;
RT "Structural and mechanistic studies on carboxymethylproline synthase
RT (CarB), a unique member of the crotonase superfamily catalyzing the first
RT step in carbapenem biosynthesis.";
RL J. Biol. Chem. 280:34956-34965(2005).
CC -!- FUNCTION: Catalyzes the formation of (2S,5S)-carboxymethylproline (t-
CC CMP) from malonyl-CoA and (S)-1-pyrroline-5-carboxylate, the first step
CC in the biosynthesis of (5R)-carbapen-2-em-3-carboxylate, a beta-lactam
CC antibiotic of the carbapenem class (PubMed:15595850, PubMed:14625287).
CC Also catalyzes the independent decarboxylation of malonyl-CoA and
CC methylmalonyl-CoA and the hydrolysis of CoA esters such as acetyl-CoA
CC and propionyl-CoA (PubMed:15595850). Catalyzes the reaction with a C2
CC epimeric mixture of methylmalonyl-CoA to give a 55:45 mixture of
CC (6R)- and (6S)-epimers of 6-methyl-t-CMP, under standard incubation
CC conditions (PubMed:21505494). {ECO:0000269|PubMed:14625287,
CC ECO:0000269|PubMed:15595850, ECO:0000269|PubMed:15726176,
CC ECO:0000269|PubMed:18972478, ECO:0000269|PubMed:21505494}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(S)-1-pyrroline-5-carboxylate + H(+) + H2O + malonyl-CoA =
CC (2S,5S)-5-carboxymethylproline + CO2 + CoA; Xref=Rhea:RHEA:36663,
CC ChEBI:CHEBI:15377, ChEBI:CHEBI:15378, ChEBI:CHEBI:16526,
CC ChEBI:CHEBI:17388, ChEBI:CHEBI:57287, ChEBI:CHEBI:57384,
CC ChEBI:CHEBI:73962; EC=2.3.1.226;
CC Evidence={ECO:0000269|PubMed:14625287, ECO:0000269|PubMed:15595850};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.0027 mM for malonyl-CoA {ECO:0000269|PubMed:15595850};
CC KM=0.0015 mM for (S)-1-pyrroline-5-carboxylate
CC {ECO:0000269|PubMed:15595850};
CC Note=kcat is 1.7 sec(-1) with malonyl-CoA as substrate. kcat is 1.52
CC sec(-1) with (S)-1-pyrroline-5-carboxylate as substrate.
CC {ECO:0000269|PubMed:15595850};
CC -!- PATHWAY: Antibiotic biosynthesis; carbapenem biosynthesis.
CC {ECO:0000305|PubMed:14625287}.
CC -!- SUBUNIT: Homotrimer. {ECO:0000269|PubMed:16096274}.
CC -!- MISCELLANEOUS: Unusual member of the enoyl-CoA hydratase/isomerase
CC family: in addition to decarboxylation and thioester hydrolysis steps,
CC catalyzes C-C bond formation leading to a substituted heterocycle. Glu-
CC 131 is important in both C-C bond formation and thioester hydrolysis
CC steps while it does not play an essential role in decarboxylation.
CC {ECO:0000269|PubMed:18972478}.
CC -!- SIMILARITY: Belongs to the enoyl-CoA hydratase/isomerase family.
CC {ECO:0000305}.
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DR EMBL; U17224; AAD38230.1; -; Genomic_DNA.
DR PDB; 2A7K; X-ray; 2.24 A; A/B/C/D/E/F/G/H/I=1-250.
DR PDB; 2A81; X-ray; 3.15 A; A/B/C=1-250.
DR PDBsum; 2A7K; -.
DR PDBsum; 2A81; -.
DR AlphaFoldDB; Q9XB60; -.
DR SMR; Q9XB60; -.
DR BioCyc; MetaCyc:MON-13571; -.
DR BRENDA; 2.3.1.226; 2140.
DR SABIO-RK; Q9XB60; -.
DR UniPathway; UPA00182; -.
DR EvolutionaryTrace; Q9XB60; -.
DR GO; GO:0016740; F:transferase activity; IEA:UniProtKB-KW.
DR GO; GO:0017000; P:antibiotic biosynthetic process; IEA:UniProtKB-KW.
DR InterPro; IPR029045; ClpP/crotonase-like_dom_sf.
DR InterPro; IPR001753; Enoyl-CoA_hydra/iso.
DR Pfam; PF00378; ECH_1; 1.
DR SUPFAM; SSF52096; SSF52096; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antibiotic biosynthesis; Transferase.
FT CHAIN 1..250
FT /note="Carboxymethylproline synthase"
FT /id="PRO_0000431235"
FT BINDING 60..64
FT /ligand="malonyl-CoA"
FT /ligand_id="ChEBI:CHEBI:57384"
FT /evidence="ECO:0000269|PubMed:16096274"
FT SITE 131
FT /note="Important for catalytic activity"
FT /evidence="ECO:0000269|PubMed:18972478"
FT MUTAGEN 79
FT /note="W->A: Forms the C6 epimers of 6-methyl-t-CMP in
FT 16:84 ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 79
FT /note="W->F: Forms the C6 epimers of 6-methyl-t-CMP in
FT 17:83 ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 108
FT /note="M->A: Forms the C6 epimers of 6-methyl-t-CMP in
FT 45:55 ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 108
FT /note="M->I: Forms the C6 epimers of 6-methyl-t-CMP in 92:8
FT ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 108
FT /note="M->L: Forms the C6 epimers of 6-methyl-t-CMP in
FT 47:53 ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 108
FT /note="M->V: Forms the C6 epimers of 6-methyl-t-CMP in 95:5
FT ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 111
FT /note="Q->N: Forms the C6 epimers of 6-methyl-t-CMP in
FT 75:25 ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT MUTAGEN 131
FT /note="E->A,Q: Does not catalyze production of (2S,5S)-5-
FT carboxymethylproline but catalyzes decarboxylation of
FT malonyl-CoA to methylmalonyl-CoA."
FT /evidence="ECO:0000269|PubMed:18972478"
FT MUTAGEN 131
FT /note="E->D: Catalyzes production of (2S,5S)-5-
FT carboxymethylproline and decarboxylation of malonyl-CoA to
FT methylmalonyl-CoA, but with much lower specific activity
FT for decarboxylation."
FT /evidence="ECO:0000269|PubMed:18972478"
FT MUTAGEN 229
FT /note="H->A: Forms the C6 epimers of 6-methyl-t-CMP in
FT 70:30 ratio of (6R):(6S) epimers."
FT /evidence="ECO:0000269|PubMed:21505494"
FT STRAND 2..7
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 10..15
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 26..41
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 47..51
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 64..68
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 73..91
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 97..101
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 103..106
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 108..113
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 116..122
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 126..128
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 131..134
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 139..149
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 151..160
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 166..171
FT /evidence="ECO:0007829|PDB:2A7K"
FT STRAND 176..179
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 181..196
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 200..228
FT /evidence="ECO:0007829|PDB:2A7K"
FT HELIX 231..237
FT /evidence="ECO:0007829|PDB:2A7K"
SQ SEQUENCE 250 AA; 27575 MW; 992F5F0829A91EAE CRC64;
MVFEENSDEV RVITLDHPNK HNPFSRTLET SVKDALARAN ADDSVRAVVV YGGAERSFSA
GGDFNEVKQL SRSEDIEEWI DRVIDLYQAV LNVNKPTIAA VDGYAIGMGF QFALMFDQRL
MASTANFVMP ELKHGIGCSV GAAILGFTHG FSTMQEIIYQ CQSLDAPRCV DYRLVNQVVE
SSALLDAAIT QAHVMASYPA SAFINTKRAV NKPFIHLLEQ TRDASKAVHK AAFQARDAQG
HFKNVLGKKY
