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Information on EC 6.3.3.6 - carbapenam-3-carboxylate synthase
for references in articles please use BRENDA:EC6.3.3.6
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EC Tree 6 Ligases
6.3 Forming carbon-nitrogen bonds
6.3.3 Cyclo-ligases
6.3.3.6 carbapenam-3-carboxylate synthase
IUBMB Comments
The enzyme is involved in the biosynthesis of the carbapenem β-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate in the bacterium Pectobacterium carotovorum.
The enzyme appears in viruses and cellular organisms
- 6.3.3.6
-
carbapenams
-
beta-lactamization
-
simplest
-
monocyclic
- crotonase
- malonyl-coa
-
bicyclic
-
stereoselective
-
beta-ls
- asparagine
-
ph-rate
-
biocatalytic
- synthases
-
viscosity
- synthesis
Reaction Schemes
showSynonyms
carbapenam synthetase, carbapenam-3-carboxylate synthase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CarA
carbapenam synthetase
CPS
EC 6.3.1.16
-
-
formerly
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + (2S,5S)-5-carboxymethylproline = AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
MetaCyc
(5R)-carbapenem carboxylate biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
6-methyl-(2S,5S)-5-carboxymethylproline cyclo-ligase (AMP-forming)
The enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate in the bacterium Pectobacterium carotovorum.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(6R)-6-ethyl-t-dimethyl-t-carboxymethylproline
AMP + (3S,5S,6R)-6-ethyl-carbapenam 3-carboxylate
-
Substrates: 75% conversion
Products: -
Products: -
?
ATP + (2R,5R)-5-carboxymethylproline
?
Substrates: low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
Products: -
Products: -
?
ATP + (2S,5R)-5-carboxymethylproline
?
Substrates: low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
Products: -
Products: -
?
ATP + (2S,6R)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (3S,5S,6R)-3,6-dimethylcabapenam-3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (2S,6S)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2S,3S,5S,6S)-3,6-dimethylcabapenam-3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (3R,6R)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2R,3S,5S,6R)-3,6-dimethylcabapenam-3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (3R,6S)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2R,3S,5S,6S)-3,6-dimethylcabapenam-3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (3S,6R)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2S,3S,5S,6R)-3,6-dimethylcabapenam-3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (4R,6R)-4,6-dimethyl-t-carboxymethylproline
AMP + (1R,3S,5S,6R)-1,6-dimethyl-carbapenam 3-carboxylate
-
Substrates: 70% conversion
Products: -
Products: -
?
ATP + (4S)-4,6,6-trimethyl-t-carboxymethylproline
AMP + (1S,3S,5S)-1,6,6-trimethyl-carbapenam 3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (4S,6R)-4,6-dimethyl-t-carboxymethylproline
AMP + (1S,3S,5S,6R)-1,6-dimethyl-carbapenam 3-carboxylate
-
Substrates: more than 90% conversion
Products: -
Products: -
?
ATP + (4S,6R)-4-methyl-6-ethyl-t-carboxymethylproline
AMP + (1S,3S,5S,6R)-6-ethyl-1-methyl-carbapenam 3-carboxylate
-
Substrates: -
Products: -
Products: -
?
ATP + (4S,6S)-4,6-dimethyl-t-carboxymethylproline
AMP + (1S,3S,5S,6S)-1,6-dimethyl-carbapenam 3-carboxylate
-
Substrates: 65% conversion
Products: -
Products: -
?
additional information
?
-
-
Substrates: CarA prefers substrates with the (4S-) and/or (6R)-stereochemistry
Products: -
Products: -
-
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: the enzyme catalyzes the formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: the kinetic mechanism is bi-ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and diphosphate is the last product released. Low activity with (2S,5R)-5-carboxymethylproline or (2R,5R)-5-carboxymethylproline
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
Substrates: catalytic Tyr-Glu dyad is demonstrated by site-directed mutagenesis and kinetic experiments that compare the wild-type enzymes to their respective mutant proteins using pHrate profiles, 32P-incorporation experiments, solvent isotope effects, proton inventory, and viscosity variation
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
Substrates: a mechanism is proposed where the rate-limiting step is beta-lactam ring formation coupled to a protein conformational change. The role of K443 throughout the reaction is undercored
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: -
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: -
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: the enzyme catalyzes the formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: -
Products: -
Products: -
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
Substrates: -
Products: -
Products: -
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
diphosphate
the competitive inhibition exhibited by diphosphate versus ATP allows the assignment of diphosphate as the last product released
L-proline
the uncompetitive nature of the inhibition of the dead-end inhibitor, L-proline, versus ATP is consistent with a mechanism with ordered substrate binding where ATP binds first followed by (2S,5S)-carboxymethylproline
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.075
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380Q
0.27
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F
0.35
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, wild-type enzyme
0.36
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380D
0.4
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380A
0.49
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380A
0.49
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380D
0.9
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
1.7
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345A
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0051
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380A
0.0059
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380D
0.0067
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380A
0.011
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345A
0.011
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
0.02
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380Q
0.17
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F
0.26
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380D
0.36
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, wild-type enzyme
0.36
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°, wild-type enzyme
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0063
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345A
0.011
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380Q
0.012
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380D
0.012
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F/E380A
0.014
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380A
0.27
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380Q
0.63
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme Y345F
0.72
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, mutant enzyme E380D
1.04
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°C, wild-type enzyme
1.04
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25°, wild-type enzyme
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.1
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
1.3
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: ATP, Ki(slope)
1.3
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
0.17
AMP
pH 7.8, 22°C, variable substrate: ATP, Ki(intercept)
0.28
AMP
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
0.005
diphosphate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
0.012
diphosphate
pH 7.8, 22°C, variable substrate: ATP, Ki(slope)
0.019
diphosphate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
90
L-proline
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
207
L-proline
pH 7.8, 22°C, variable substrate: ATP, Ki(intercept)
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). CARA_PECCC
503
0
55998
Swiss-Prot
other Location (Reliability: 2)
Q83XP1_STRCT
458
0
48400
TrEMBL
other Location (Reliability: 1)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
55998
4 * 55998, calculated from sequence, the four monomers form a tetramer with a hole in the center, crystallographic data
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
tetramer
4 * 55998, calculated from sequence, the four monomers form a tetramer with a hole in the center, crystallographic data
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting drop method, the crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline, ATP analog alpha,beta-methyleneadenosine 5'-triphosphate, and a single Mg2+
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E380D
-
retains a functional general base with a pKa of about 7.4 for kcat. The acidic region of the log (kcat/Km) versus pH profiles displays an ionizable group with a pKa of about 7.7. kcat/Km is 1.4fold lower compared to wild-type value
E380Q
-
variant displays a plateau in the acidic region. The acidic region of the log (kcat/Km) versus pH profiles becomes pH-independent for E380Q. kcat/Km is 3.8fold lower compared to wild-type value
K443A
-
at pH 8.0, 6.33 and 9.33 the mutant enzyme shows no measurable activity
K443M
-
at pH 8.0, 6.33 and 9.33 the mutant enzyme shows no measurable activity
Y345A
-
mutant exhibits a 5fold increase in Km of (2S,5S)-5-carboxymethylproline and a 165fold decrease in specificity constant compared to wild-type enzyme. kcat/Km is 165fold lower compared to wild-type value
Y345F
-
pH-dependent kcat and kcat/Km plots retain the bell-shaped curve of wild-type CPS with similar pK values. kcat/Km is 1.7fold lower compared to wild-type value
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
overexpression of the plasmid pET24a/carA transformed in Escherichia coli BL21(DE3) cells
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
-
stereoselctive synthesis of dimethylsubstituted carbapenams using engineered 5-carboxymethylproline synthase and carbapenam-3-carboxylate synthase
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gerratana, B.; Stapon, A.; Townsend, C.A.
Inhibition and alternate substrate studies on the mechanism of carbapenam synthetase from Erwinia carotovora
Biochemistry
42
7836-7847
2003
Pectobacterium carotovorum (Q9XB61), Pectobacterium carotovorum
brenda
Arnett, S.O.; Gerratana, B.; Townsend, C.A.
Rate-limiting steps and role of active site Lys443 in the mechanism of carbapenam synthetase
Biochemistry
46
9337-9345
2007
Pectobacterium carotovorum
brenda
Raber, M.L.; Arnett, S.O.; Townsend, C.A.
A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase
Biochemistry
48
4959-40971
2009
Pectobacterium carotovorum
brenda
Miller, M.T.; Gerratana, B.; Stapon, A.; Townsend, C.A.; Rosenzweig, A.C.
Crystal structure of carbapenam synthetase (CarA)
J. Biol. Chem.
278
40996-41002
2003
Pectobacterium carotovorum (Q9XB61)
brenda
Tahlan, K.; Jensen, S.
Origins of the beta-lactam rings in natural products
J. Antibiot.
66
401-410
2013
Streptantibioticus cattleyicolor (Q83XP1), Pectobacterium carotovorum (Q9XB61)
brenda
Hamed, R.; Henry, L.; Claridge, T.; Schofield, C.
Stereoselective production of dimethyl-substituted carbapenams via engineered carbapenem biosynthesis enzymes
ACS Catal.
7
1279-1285
2017
Pectobacterium carotovorum
-
brenda
Hamed, R.B.; Gomez-Castellanos, J.R.; Henry, L.; Warhaut, S.; Claridge, T.D.W.; Schofield, C.J.
Biocatalytic production of bicyclic beta-lactams with three contiguous chiral centres using engineered crotonases
Commun. Chem.
2
007
2019
Pectobacterium carotovorum
brenda
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