Information on EC 2.3.1.226 - carboxymethylproline synthase

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The expected taxonomic range for this enzyme is: Pectobacterium carotovorum

EC NUMBER
COMMENTARY hide
2.3.1.226
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RECOMMENDED NAME
GeneOntology No.
carboxymethylproline synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
malonyl-CoA + (S)-1-pyrroline-5-carboxylate + H2O = CoA + (2S,5S)-5-carboxymethylproline + CO2
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(5R)-carbapenem carboxylate biosynthesis
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Carbapenem biosynthesis
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
malonyl-CoA:(S)-1-pyrroline-5-carboxylate malonyltransferase (cyclizing)
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
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ethylmalonyl-CoA + (2S,5S)-carboxymethylproline + CO2
CoA + (2S,5S,6R)-6-ethyl-5-carboxymethylproline
show the reaction diagram
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the substrate is an equilibrium mixture of L-glutamate semialdehyde, 5-hydroxy-L-proline and L-pyrroline-5-carboxylate. Some of the CarB variants catalyse the production of the two C6 epimers of (2S,5S)-6-ethyl-5-carboxymethylproline
wild-type enzyme produces a 65:35 mixture of (6R)- and (6S)-epimer
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?
ethylmalonyl-CoA + (2S,5S)-carboxymethylproline + CO2
CoA + (2S,5S,6S)-6-ethyl-5-carboxymethylproline
show the reaction diagram
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the substrate is an equilibrium mixture of L-glutamate semialdehyde, 5-hydroxy-L-proline and L-pyrroline-5-carboxylate. Some of the CarB variants catalyse the production of the two C6 epimers of (2S,5S)-6-ethyl-5-carboxymethylproline
wild-type enzyme produces a 65:35 mixture of (6R)- and (6S)-epimer
-
?
ethylmalonyl-CoA + L-2-aminoadipate semialdehyde
CoA + (2S,6S,7R)-7-ethyl-carboxymethylpipecolic acid
show the reaction diagram
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activity with mutant enzymes W79F, W79F/M108A and W79F/M108V. mutant W79A: epimeric ethylmalonyl–CoA produces solely (2S,6S,7S)-(2S,6S)-carboxymethylpipecolic acid via reaction of (2R)-ethylmalonyl-CoA, whereas incubation of (2S)-ethylmalonyl–CoA and L-2-aminoadipate semialdehyde results in no detectable production of 7-ethyl-(2S,6S)-carboxymethylpipecolic acid
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-
?
ethylmalonyl-CoA + L-2-aminoadipate semialdehyde
CoA + (2S,6S,7S)-7-ethyl-carboxymethylpipecolic acid
show the reaction diagram
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activity with mutant enzymes W79F, W79F/M108A and W79F/M108V. mutant W79A: epimeric ethylmalonyl–CoA produces solely (2S,6S,7S)-(2S,6S)-carboxymethylpipecolic acid via reaction of (2R)-ethylmalonyl-CoA, whereas incubation of (2S)-ethylmalonyl–CoA and L-2-aminoadipate semialdehyde results in no detectable production of 7-ethyl-(2S,6S)-carboxymethylpipecolic acid
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-
?
malonyl-CoA + (S)-1-pyrroline-5-carboxylate + H2O
CoA + (2S,5S)-5-carboxymethylproline + CO2
show the reaction diagram
malonyl-CoA + (S)-1-pyrroline-5-carboxylate + H2O
CoA + (2S,5S)-carboxymethylproline + CO2
show the reaction diagram
malonyl-CoA + L-2-aminopimelate semialdehyde
CoA + (2S,7S)-7-(carboxymethyl)-azepane-2-carboxylic acid + CO2
show the reaction diagram
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the CarB His229Ala variant is able to form the target compound in a yield of about 3 times higher than that obtained with wild-type CarB
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?
malonyl-CoA + L-glutamate-gamma-semialdehyde
CoA + (2S,5S)-5-carboxymethylproline + CO2
show the reaction diagram
methylmalonyl-CoA + (2S,5S)-5-carboxymethylproline + CO2
CoA + (2S,5S,6R)-6-methyl-5-carboxymethylproline
show the reaction diagram
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the substrate is an equilibrium mixture of L-glutamate semialdehyde, 5-hydroxy-L-proline and L-pyrroline-5-carboxylate
wild-type enzyme produces a 55:45 mixture of (6R)- and (6S)-epimer
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?
methylmalonyl-CoA + (2S,5S)-5-carboxymethylproline + CO2
CoA + (2S,5S,6S)-6-methyl-5-carboxymethylproline
show the reaction diagram
-
the substrate is an equilibrium mixture of L-glutamate semialdehyde, 5-hydroxy-L-proline and L-pyrroline-5-carboxylate
wild-type enzyme produces a 55:45 mixture of (6R)- and (6S)-epimer
-
?
methylmalonyl-CoA + (S)-1-pyrroline-5-carboxylate + H2O
CoA + (2S,5S)-6-methyl-5-carboxymethylproline + CO2
show the reaction diagram
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-
-
-
?
methylmalonyl-CoA + L-2-aminoadipate semialdehyde
CoA + (2S,6S,7R)-7-methyl-carboxymethylpipecolic acid
show the reaction diagram
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wild-type enzyme produces a 66:34 mixture of (7R)- and (7S)-epimer
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?
methylmalonyl-CoA + L-2-aminoadipate semialdehyde
CoA + (2S,6S,7S)-7-methyl-carboxymethylpipecolic acid
show the reaction diagram
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wild-type enzyme produces a 66:34 mixture of (7R)- and (7S)-epimer
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?
additional information
?
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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
malonyl-CoA + (S)-1-pyrroline-5-carboxylate + H2O
CoA + (2S,5S)-5-carboxymethylproline + CO2
show the reaction diagram
malonyl-CoA + (S)-1-pyrroline-5-carboxylate + H2O
CoA + (2S,5S)-carboxymethylproline + CO2
show the reaction diagram
malonyl-CoA + L-glutamate-gamma-semialdehyde
CoA + (2S,5S)-5-carboxymethylproline + CO2
show the reaction diagram
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?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0015
(S)-1-pyrroline-5-carboxylate
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pH 7.8, 22°C
0.0027
malonyl-CoA
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pH 7.8, 22°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.52
(S)-1-pyrroline-5-carboxylate
Pectobacterium carotovorum
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pH 7.8, 22°C
1.7
malonyl-CoA
Pectobacterium carotovorum
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pH 7.8, 22°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
984
(S)-1-pyrroline-5-carboxylate
Pectobacterium carotovorum
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pH 7.8, 22°C
3430
630
malonyl-CoA
Pectobacterium carotovorum
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pH 7.8, 22°C
76
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
trimer
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3 * 27575, calculated from sequence, negative ion electrospray MS, enzyme exists predominantly in a trimeric form
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, crystal structure of CarB in the presence and absence of acetyl-CoA at 2.24 A and 3.15 A resolution. The structures reveal that CarB contains a conserved oxy-anion hole probably required for decarboxylation of malonyl-CoA and stabilization of the resultant enolate
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
M108A
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 45:55 compared to wild-type ratio of 55:45
M108I
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 92:8 compared to wild-type ratio of 55:45
M108L
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 47:53 compared to wild-type ratio of 55:45. Forms the C6 epimers (6R):(6S) of (2S,5S)-6-ethyl-5-carboxymethylproline in the ratio of 72:18 compared to wild-type ratio of 65:35. Forms the C7 epimers (7R):(7S) of 7-methyl-(2S,6S)-carboxymethylpipecolic acid in the ratio of 60:40 compared to the wild-type ration of 66:34
Q111N
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 70:30 compared to wild-type ratio of 55:45
W79A
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 16:84 compared to wild-type ratio of 55:45. Forms the C6 epimers (6R):(6S) of (2S,5S)-6-ethyl-5-carboxymethylproline in the ratio of 12:88 compared to wild-type ratio of 65:35. Forms the C7 epimers (7R):(7S) of 7-methyl-(2S,6S)-carboxymethylpipecolic acid in the ratio of 1:99 compared to the wild-type ration of 66:34
W79F/M108A
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 11:89 compared to wild-type ratio of 55:45. Forms the C6 epimers (6R):(6S) of (2S,5S)-6-ethyl-5-carboxymethylproline in the ratio of 17:83 compared to wild-type ratio of 65:35. Forms the C7 epimers (7R):(7S) of 7-methyl-(2S,6S)-carboxymethylpipecolic acid in the ratio of 5:95 compared to the wild-type ration of 66:34
W79F/M108V
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forms the C6 epimers (6R):(6S) of (2S,5S)-6-methyl-5-carboxymethylproline in the ratio of 56:44 compared to wild-type ratio of 55:45. Forms the C6 epimers (6R):(6S) of (2S,5S)-6-ethyl-5-carboxymethylproline in the ratio of 34:66 compared to wild-type ratio of 65:35. Forms the C7 epimers (7R):(7S) of 7-methyl-(2S,6S)-carboxymethylpipecolic acid in the ratio of 24:76 compared to the wild-type ration of 66:34
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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carboxymethylproline synthases is a biocatalysts for preparing functionalised N-heterocycles in a diastereoselective fashion. The products can be converted into the respective bicyclic beta-lactams of potential application in the semisynthesis of stable beta-lactam antibiotics
synthesis