A Minimalistic Bioorganic Approach to Identify Substrate Binding Features in Metallo-B-Lactamase

POEYLAUT PALENA, ANDRÉS A.; TOMATIS, PABLO E.; MATA, ERNESTO G. AND VILA, ALEJANDRO J.

Rosario, Argentina

Congreso; SAB XXXV Annual Meeting; 2006

Metallo­–b–Lactamases (MβLs) are responsible for one of the ultimate kind of clinical microbiological resistance towards b–lactam antibiotics. They are zinc-dependent enzymes able to hydrolyze a broad range of b-lactam antibiotics, while useful clinical inhibitor against them is still lacking. Their active sites are able to bind up to two Zn(II) ions. How the substrates bind to MβLs active site is still not clear. Some clues about the mode of binding are given by X–Ray structures of “enzymes-product” complexes. The only common structure shared among the good substrates for this enzymes is the b–lactam ring (2–azetidinone) and a carboxylate in a position to the b–lactam nitrogen. Despite their broad substrate spectrum, MβLs fail to hydrolyze monocyclic b–lactams such as aztreonam. This could be related to the structural variation of aztreonam compared with the other b–lactam compounds. Because of aztreonam is susceptible to non–enzymatic hydrolysis in the same extent that the other b–lactam antibiotics, a possible lack of binding is argued.To identify (1) the minimal elements required for allowing efficient binding and chemical events and (2) the chemical features that make aztreonam different to current MβL substrates, we synthetized sodium (2–oxo–azetidinyl)–acetate. This compound shares a monocyclic structure with aztreonam, and bears the a carboxylate conserve in most clinically useful antibiotic but lacking in aztreonam. In this work we analyze the binding properties and the possible substrate–like behavior of sodium (2–oxo–azetidinyl)–acetate and aztreonam to the Bacillus cereus MβL BcII. Binding to the active site was studied by different techniques such as stopped flow fluorescence quenching, NMR spectroscopy, paramagnetic NMR and UV–visible spectroscopy of cobalt(II) substituted BcII, and competitive inhibition of substrate hydrolysis.