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,βmethyleneadenosine 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/PP_iMg²⁺ has been determined. The CMA-AMP/PP_i/Mg²⁺ and DGPC·AMP/PP_i/Mg²⁺ DGPC·AMp/PP_i/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.