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Information on EC 6.3.3.6 - carbapenam-3-carboxylate synthase
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6.3.3.6 carbapenam-3-carboxylate synthaseIUBMB Comments
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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Word Map
- 6.3.3.6
-
carbapenams
-
beta-lactamization
- crotonase
-
simplest
-
monocyclic
- synthases
- asparagine
-
stereoselective
- malonyl-coa
-
viscosity
-
ph-rate
-
biocatalytic
-
beta-ls
-
bicyclic
- synthesis
The enzyme appears in viruses and cellular organisms
Reaction Schemes
Synonyms
CarA, carbapenam 3-carboxylate synthase, carbapenam synthetase, CPS, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CarA
carbapenam synthetase
CPS
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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
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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
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + (2R,5R)-5-carboxymethylproline
?
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
-
-
?
ATP + (2S,5R)-5-carboxymethylproline
?
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
-
-
?
ATP + (2S,6R)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (3S,5S,6R)-3,6-dimethylcabapenam-3-carboxylate
-
-
-
-
?
ATP + (2S,6S)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2S,3S,5S,6S)-3,6-dimethylcabapenam-3-carboxylate
-
-
-
-
?
ATP + (3R,6R)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2R,3S,5S,6R)-3,6-dimethylcabapenam-3-carboxylate
-
-
-
-
?
ATP + (3R,6S)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2R,3S,5S,6S)-3,6-dimethylcabapenam-3-carboxylate
-
-
-
-
?
ATP + (3S,6R)-2,6-dimethyl-t-carboxymethylproline
AMP + diphosphate + (2S,3S,5S,6R)-3,6-dimethylcabapenam-3-carboxylate
-
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
the enzyme catalyzes the formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
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
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
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 pHĆ¢ĀĀrate profiles, 32P-incorporation experiments, solvent isotope effects, proton inventory, and viscosity variation
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
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
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
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
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
the enzyme catalyzes the formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
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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, 25ĆĀ°C, mutant enzyme E380Q
0.27
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F
0.35
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, wild-type enzyme
0.36
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380D
0.4
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380A
0.49
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380A
0.49
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380D
0.9
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380Q
1.7
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°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, 25ĆĀ°C, mutant enzyme Y345F/E380A
0.0059
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380D
0.0067
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380A
0.011
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345A
0.011
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380Q
0.02
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380Q
0.17
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F
0.26
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380D
0.36
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, wild-type enzyme
0.36
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°, 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, 25ĆĀ°C, mutant enzyme Y345A
0.011
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380Q
0.012
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380A
0.012
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F/E380D
0.014
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380A
0.27
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380Q
0.63
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme Y345F
0.72
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, mutant enzyme E380D
1.04
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°C, wild-type enzyme
1.04
(2S,5S)-5-carboxymethylproline
-
pH 8.0, 25ĆĀ°, 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, 22ĆĀ°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
1.3
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22ĆĀ°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
1.3
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22ĆĀ°C, variable substrate: ATP, Ki(slope)
0.17
AMP
pH 7.8, 22ĆĀ°C, variable substrate: ATP, Ki(intercept)
0.28
AMP
pH 7.8, 22ĆĀ°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
0.005
diphosphate
pH 7.8, 22ĆĀ°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
0.012
diphosphate
pH 7.8, 22ĆĀ°C, variable substrate: ATP, Ki(slope)
0.019
diphosphate
pH 7.8, 22ĆĀ°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
90
L-proline
pH 7.8, 22ĆĀ°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
207
L-proline
pH 7.8, 22ĆĀ°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
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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
-
A0A1V5M191_9FIRM
460
0
52893
TrEMBL
-
A0A7W8WQW9_9BURK
496
0
54533
TrEMBL
-
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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
Streptomyces cattleya (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
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