Crystal Structure of Carbapenam Synthetase (CarA)

J Biol Chem. 2003 Oct 17;278(42):40996-1002. doi: 10.1074/jbc.M307901200. Epub 2003 Jul 30.


Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-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 alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-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 beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-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 Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Amino Acid Sequence
  • Anti-Bacterial Agents / chemistry*
  • Binding Sites
  • Carbapenems / chemistry
  • Carbapenems / metabolism*
  • Carbon-Nitrogen Ligases / chemistry*
  • Conserved Sequence
  • Crystallography, X-Ray
  • Escherichia coli / metabolism
  • Glutaminase / chemistry
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary


  • Anti-Bacterial Agents
  • Carbapenems
  • Adenosine Triphosphate
  • Glutaminase
  • Carbon-Nitrogen Ligases
  • carbapenam synthetase

Associated data

  • PDB/1Q15
  • PDB/1Q19