Structural studies of the beta-lactam ring-forming enzymes beta-lactam synthetase and carbapenam synthetase.

摘要

β-lactam antibiotics are an essential component of modern medicine; however bacterial resistance caused by β-lactamases has compromised their effectiveness. Compounds have been discovered, such as clavulanic acid produced by the bacteria Streptomyces clavuligerus, that can combat bacterial resistance by potently blocking β-lactamases. When clavulanic acid is used in conjunction with β-lactam antibiotics that are susceptible to β-lactamases, the antibiotics effectiveness is restored.; The bicyclic nucleus of clavulanic acid is chemically very similar to the antibiotics it protects; yet in some cases the biosynthetic pathway that forms clavulanic acid is entirely different. The β-lactam ring of clavulanic acid is formed by the enzyme β-lactam synthetase (β-LS) in the second step of a eight step biosynthetic pathway.; The objective of this thesis has been to observe β-LS through a series of X-ray crystallographic snapshots in order to determine more about its chemical mechanism. The structures of the apo, ATP/Mg2+ bound, and substrate N2-(carboxyethyl)-L-arginine (CEA) • ATP analog, α,β-methyleneadenosine 5′ -triphosphate (AMP-CPP)/Mg2+, illuminate changes in active site geometry that favor adenylation. In addition, an acyladenylate intermediate has been trapped. The crystals soaked with the substrate analog N2-(carboxymethyl)-L-arginine (CMA) and ATP produced CMA-adenylate, which represents a close structural analog of the previously proposed CEA-adenylate intermediate. Finally, the structure of the bound ternary product complex, deoxyguanidinoproclavaminic acid (DGPC)/AMP/PPi/Mg 2+, was determined. The CMA-AMP/PPi/Mg2+ and DGPC/AMP/PPi/Mg 2+ structures reveal interactions in the active site that facilitate β-lactam formation. All of these structures contained Mg2+, which plays a critical role in both the adenylation and β-lactamization reactions.; The structure of a homologous protein carabapenam synthetase (CS), which catalyzes a very similar reaction in the biosynthesis of the β-lactam antibiotic (5R)-carbapen-2-em-3-carboxylic acid, has also been solved. CS has considerable structural homology to β-LS including many key active site residues. The structure also serves to reinforce the mechanistic conclusions drawn from the β-LS structures.