Crystal structure of carbapenam synthetase (CarA).
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.
Miller
Matthew T
MT
Department of Biochemistry, Northwestern University, Evanston, Illinois 60208, USA.
Gerratana
Barbara
B
Stapon
Anthony
A
Townsend
Craig A
CA
Rosenzweig
Amy C
AC
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001665:Binding Sites; D015780:Carbapenems / Q000737:chemistry / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005972:Glutaminase / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D000255:Adenosine Triphosphate; D005972:Glutaminase; D019731:Carbon-Nitrogen Ligases; C479139:carbapenam synthetase