Information on EC 6.3.3.6 - carbapenam-3-carboxylate synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
6.3.3.6
-
RECOMMENDED NAME
GeneOntology No.
carbapenam-3-carboxylate synthase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + (2S,5S)-5-carboxymethylproline = AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
show the reaction diagram
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(5R)-carbapenem carboxylate biosynthesis
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Biosynthesis of antibiotics
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Carbapenem biosynthesis
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(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.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + (2R,5R)-5-carboxymethylproline
?
show the reaction diagram
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
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-
?
ATP + (2S,5R)-5-carboxymethylproline
?
show the reaction diagram
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + (2S,5S)-5-carboxymethylproline
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
activity depends on
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3S,5S)-carbapenam 3-carboxylate
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AMP
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uncompetitive inhibition versus both substrates
diphosphate
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the competitive inhibition exhibited by diphosphate versus ATP allows the assignment of diphosphate as the last product released
L-proline
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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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6
(2R,5R)-5-carboxymethylproline
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pH 7.8, 22°C
7
(2S,5R)-5-carboxymethylproline
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pH 7.8, 22°C
0.075 - 1.7
(2S,5S)-5-carboxymethylproline
0.11
ATP
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pH 7.8, 22°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.122
(2R,5R)-5-carboxymethylproline
Pectobacterium carotovorum
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pH 7.8, 22°C
0.2
(2S,5R)-5-carboxymethylproline
Pectobacterium carotovorum
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pH 7.8, 22°C
0.0051 - 0.36
(2S,5S)-5-carboxymethylproline
0.28
ATP
Pectobacterium carotovorum
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pH 7.8, 22°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.026
(2R,5R)-5-carboxymethylproline
Pectobacterium carotovorum
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pH 7.8, 22°C
42493
0.027
(2S,5R)-5-carboxymethylproline
Pectobacterium carotovorum
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pH 7.8, 22°C
42492
0.0063 - 1.2
(2S,5S)-5-carboxymethylproline
2698
2.5
ATP
Pectobacterium carotovorum
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pH 7.8, 22°C
4
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.1 - 1.3
(3S,5S)-carbapenam 3-carboxylate
0.17 - 0.28
AMP
0.005 - 0.019
diphosphate
90 - 207
L-proline
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55998
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4 * 55998, calculated from sequence, the four monomers form a tetramer with a hole in the center, crystallographic data
56000
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2 * 56000, calculated from sequence
119000
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native PAGE
235000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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2 * 56000, calculated from sequence
tetramer
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4 * 55998, calculated from sequence, the four monomers form a tetramer with a hole in the center, crystallographic data
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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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
wild-type, K443A and K443M mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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overexpression of the plasmid pET24a/carA transformed in Escherichia coli BL21(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E380A
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kcat/Km is 74.3fold lower compared to wild-type value
E380D
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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
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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
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at pH 8.0, 6.33 and 9.33 the mutant enzyme shows no measurable activity
K443M
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at pH 8.0, 6.33 and 9.33 the mutant enzyme shows no measurable activity
Y345A
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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
Y345A/E380A
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kcat/Km is 87fold lower compared to wild-type value
Y345F
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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
Y345F/E380D
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kcat/Km is 87fold lower compared to wild-type value
Y345F/E380Q
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kcat/Km is 95fold lower compared to wild-type value