The Catalytic Cycle of β-Lactam Synthetase Observed by X-Ray Crystallographic Snapshots

The catalytic cycle of the ATP/Mg2+-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/Mg2+and N2-(carboxyethyl)-L-arginine (CEA) to the apoenzyme. The apo and ATP/Mg2+structures described here, along with the previously described CEA·α,β-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 N2-(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/PPi/Mg2+has been determined. The CMA-AMP/PPi/Mg2+and DGPC· AMP/PPi/Mg2+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/Mg2+structure in that two Mg2+ions are found in the active sites. These Mg2+ions play critical roles in both the adenylation and β-lactamization reactions.