Crystal Structure of Carbapenam Synthetase (CarA)

Matthew T. Miller, Barbara Gerratana, Anthony Stapon, Craig A. Townsend, Amy C. Rosenzweig*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the β-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to β-lactam synthetase (β-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and β-LS mediate substrate adenylation followed by β-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg 2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog α,β -methylene-adenosine 5′-triphosphate (AMP-CPP), and a single Mg 2+ ion have been determined. CarA forms a tetramer. Each monomer resembles β-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in β-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in β-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg 2+ coordination is also different. Several key residues in the active site are conserved between CarA and β-LS, supporting proposed roles in β-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics.

Original languageEnglish (US)
Pages (from-to)40996-41002
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number42
DOIs
StatePublished - Oct 17 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Crystal Structure of Carbapenam Synthetase (CarA)'. Together they form a unique fingerprint.

Cite this