The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

The catalytic cycle of the ATP/Mg(2+)-dependent enzyme β-lactam synthetase (β-LS) from Streptomyces clavuligerus has been observed through a series of x-ray crystallographic snapshots. Chemistry is initiated by the ordered binding of ATP/Mg(2+) and N(2)-(carboxyethyl)-l-arginine (CEA) to the apoenzyme. The apo and ATP/Mg(2+) structures described here, along with the previously described CEA⋅α,β-methyleneadenosine 5′-triphosphate (CEA⋅AMP-CPP)/Mg(2+) structure, illuminate changes in active site geometry that favor adenylation. In addition, an acyladenylate intermediate has been trapped. The substrate analog N(2)-(carboxymethyl)-l-arginine (CMA) was adenylated by ATP in the crystal and represents a close structural analog of the previously proposed CEA-adenylate intermediate. Finally, the structure of the ternary product complex deoxyguanidinoproclavaminic acid (DGPC)⋅AMP/PP(i)/Mg(2+) has been determined. The CMA-AMP/PP(i)/Mg(2+) and DGPC⋅AMP/PP(i)/Mg(2+) structures reveal interactions in the active site that facilitate β-lactam formation. All of the ATP-bound structures differ from the previously described CEA⋅AMP-CPP/Mg(2+) structure in that two Mg(2+) ions are found in the active sites. These Mg(2+) ions play critical roles in both the adenylation and β-lactamization reactions.