|Gene:||carA||Accession Number: G-10343 (MetaCyc)|
Subunit composition of
carbapenam synthetase = [CarA]4
carbapenam synthetase subunit = CarA
The enzyme carbapenam synthetase catalyzes formation of the β-lactam ring in a reaction analogous to β-lactam synthase, and shows amino acid sequence homology to this enzyme. The enzyme is ATP and Mg2+-dependent. Kinetic analysis suggested that ATP binds to the enzyme first, then (2S,5S)-5-carboxymethyl proline. An acyladenylate intermediate is formed, followed by formation of the β-lactam ring by intramolecular amide bond formation. Recombinant carbapenam synthetase has been produced in Escherichia coli and purified [Gerratana03, Sleeman04].
Each monomer of the homotetramer contains two domains with the second, C-terminal domain containing the active site. The enzyme can use a range of substrates, suggesting its potential use in engineering novel carbapenem antibiotics. The crystal structure has been determined [Miller03b]. Reviewed in [Coulthurst05]
Molecular Weight of Polypeptide: 55.998 kD (from nucleotide sequence)
Relationship Links: Entrez-Nucleotide:RELATED-TO:U17224 , InterPro:IN-FAMILY:IPR001962 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR015230 , InterPro:IN-FAMILY:IPR029055 , PDB:Structure:1q15 , PDB:Structure:1Q15 , PDB:Structure:1Q19 , Pfam:IN-FAMILY:PF00733 , Pfam:IN-FAMILY:PF09147
Enzymatic reaction of: carbapenam synthetase
EC Number: 18.104.22.168
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.
The reaction is physiologically favored in the direction shown.
Alternative Substrates for (2S,5S)-5-carboxymethyl proline: (2R,5R)-5-carboxymethyl proline [Gerratana03 ] , (2S,5S)-5-carboxymethyl proline [Gerratana03 ] , (2S,5S)-5-carboxyethyl proline [Gerratana03 ] , (2S,5R)-5-carboxymethyl proline [Gerratana03 ] , D,L-aminopimelate [Gerratana03 ] , D-aminoadipate [Gerratana03 ] , L-2-aminoadipate [Gerratana03 ] , L-glutamate [Gerratana03 ]
In Pathways: (5R)-carbapenem carboxylate biosynthesis
The steady state kinetic mechanism of this enzyme has been characterized. Studies of its reactions with the diacids glutarate, adipate and pimelate as alternate substrates showed that the reaction stopped at an acyladenylate intermediate, supporting the formation of this intermediate in the reaction mechanism. Compounds 5-aminopentanoate and 6-aminohexanoate which lack the α-carboxylate groups of aminoadipate and aminopimelate were not substrates, and were very weak competitive inhibitors. In addition, L-aspartate, succinate and 3-(carboxymethyl-amino)-propanoate were not substrates. [Gerratana03]
Inhibitor AMP was uncompetitive with respect to both ATP and (2S,5S)-5-carboxymethyl proline; diphosphate was competitive with respect to ATP and noncompetitive with respect to (2S,5S)-5-carboxymethyl proline; (3S,5S)-carbapenam-3-carboxylate was uncompetitive with respect to (2S,5S)-5-carboxymethyl proline and noncompetitive with respect to ATP; and L-proline was uncompetitive with respect to ATP and competitive with respect to (2S,5S)-5-carboxymethyl proline. [Gerratana03]
Gerratana03: Gerratana B, Stapon A, Townsend CA (2003). "Inhibition and alternate substrate studies on the mechanism of carbapenam synthetase from Erwinia carotovora." Biochemistry 42(25);7836-47. PMID: 12820893
McGowan05: McGowan SJ, Barnard AM, Bosgelmez G, Sebaihia M, Simpson NJ, Thomson NR, Todd DE, Welch M, Whitehead NA, Salmond GP (2005). "Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway." Mol Microbiol 55(2);526-45. PMID: 15659168
McGowan96: McGowan SJ, Sebaihia M, Porter LE, Stewart GS, Williams P, Bycroft BW, Salmond GP (1996). "Analysis of bacterial carbapenem antibiotic production genes reveals a novel beta-lactam biosynthesis pathway." Mol Microbiol 22(3);415-26. PMID: 8939426
Sleeman04: Sleeman MC, Schofield CJ (2004). "Carboxymethylproline synthase (CarB), an unusual carbon-carbon bond-forming enzyme of the crotonase superfamily involved in carbapenem biosynthesis." J Biol Chem 279(8);6730-6. PMID: 14625287
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