Abstract
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.
Highlights
The carbapenem class of -lactam antibiotics exhibits a broad spectrum of activity and is resistant to inactivation by -lactamases (1)
Carbapenam synthetase (CarA) is homologous to -lactam synthetase (LS), which is involved in clavulanic acid biosynthesis
A detailed understanding of carbapenem biosynthesis is crucial to developing improved production procedures as well as new antibiotics
Summary
Protein Overexpression and Purification—CarA for initial crystallization trials and soaking experiments was expressed as described previously (12). To generate selenomethionine-substituted protein, the expression vector pET24a/carA (10) was transformed into Escherichia coli strain B834(DE3) (Novagen) Colonies from this transformation were used to inoculate two 5-ml tubes of LB medium containing 50 g/ml kanamycin. CarA-containing fractions were identified by SDS-PAGE, pooled, applied in 5-ml aliquots to a 175-ml Superdex 200 column (Amersham Biosciences), and eluted at 1 ml/min with 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, and 1 mM DTT. The optimized precipitant solution for selenomethionine-substituted protein consisted of 30% (w/v) PEG 3350, 15% (v/v) glycerol, and 100 mM sodium citrate, pH 5.3 For data collection, these crystals were transferred to a cryosolution composed of 30% (w/v) PEG 3350, 25 mM HEPES, pH 7.5, 50 mM sodium citrate, pH 5.3, 15% (v/v) glycerol, and 250 mM NaCl. After a 1–5-min soak, the crystals were flash-cooled in liquid nitrogen. Wavelength (Å) Resolution range (Å) Unique observations Total observations Completenessa (%) Rsymb I/ Figure of meritc
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