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

The catalytic cycle of the ATP/Mg2+-dependent enzyme beta-lactam synthetase (beta-LS) from Streptomyces clavuligerus has been observed through a series of x-ray crystallographic snapshots. Chemistry is initiated by the ordered binding of ATP/Mg2+ and N-2-(carboxyethyl)-L-arginine (CEA) to the apoenzyme. The apo and ATP/Mg2+ structures described here, along with the previously described CEA(.)alpha,beta-methyleneadenosine 5'-triphosphate (CEA(.)AMP-CPP)/Mg2+ 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 deoxyguaniclinoproclavaminic acid (DGPC)(.)AMP/PPi/Mg2+ has been determined. The CMA-AMP/PPi/Mg2+ and DGPC(.)AMP/PPi/Mg2+ structures reveal interactions in the active site that facilitate P-lactam formation. All of the ATP-bound structures differ from the previously described CEA(.)AMP-Cpp/Mg2+ structure in that two Mg2+ ions are found in the active sites. These Mg2+ ions play critical roles in both the adenylation and beta-lactamization reactions.