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/PPiMg2+ has been determined. The CMA-AMP/PPi/Mg2+ and DGPC·AMP/PPi/Mg2+ 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.
|Original language||English (US)|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Nov 12 2002|
ASJC Scopus subject areas