MetaCyc Enzyme: carbapenam synthetase

Gene: carA Accession Number: G-10343 (MetaCyc)

Species: Pectobacterium carotovorum carotovorum

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 [Miller03a]. Reviewed in [Coulthurst05]

Gene Citations: [McGowan05, McGowan96, Li00]

Molecular Weight of Polypeptide: 55.998 kD (from nucleotide sequence)

Unification Links: Protein Model Portal:Q9XB61 , SMR:Q9XB61 , UniProt:Q9XB61

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

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Created 30-Nov-2007 by Fulcher CA , SRI International

Enzymatic reaction of: carbapenam synthetase

Synonyms: CarA

EC Number:

(2S,5S)-5-carboxymethyl proline + ATP <=> (3S,5S)-carbapenam-3-carboxylate + AMP + diphosphate + H+

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]

Cofactors or Prosthetic Groups: Mg2+ [Gerratana03], ATP [Gerratana03]

Inhibitors (Uncompetitive): AMP [Gerratana03]

Inhibitors (Unknown Mechanism): diphosphate [Gerratana03] , L-proline [Gerratana03] , (3S,5S)-carbapenam-3-carboxylate [Gerratana03]


Coulthurst05: Coulthurst SJ, Barnard AM, Salmond GP (2005). "Regulation and biosynthesis of carbapenem antibiotics in bacteria." Nat Rev Microbiol 3(4);295-306. PMID: 15759042

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

Li00: Li R, Stapon A, Blanchfield JT, Townsend CA (2000). "Three unusual reactions mediate carbapenem and carbapenam biosynthesis." J. Am. Chem. Soc. 122, 9296-9297.

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

Miller03a: Miller MT, Gerratana B, Stapon A, Townsend CA, Rosenzweig AC (2003). "Crystal structure of carbapenam synthetase (CarA)." J Biol Chem 278(42);40996-1002. PMID: 12890666

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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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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