Relevance of a conserved Asp residue to metallo-beta-lactamase activity

LLARRULL, L.I.; VILA, A.J.

Buenos Aires

Congreso; XIV International Biophysics Congress; 2002

SAB - IUPAB

Metallo-beta-lactamases are enzymes with a zinc binding motif in their active site, that is essential for activity. They display a strong hydrolytic ability towards different antibiotics, specially carbapenems. Since clinically useful inhibitors are ineffective against metallo-beta-lactamases, a rational design of new inhibitors should necessarily rely on a thorough knowledge of their catalytic mechanism. Asp90 is conserved in all known metallo-beta-lactamases, and is involved in zinc coordination in enzymes containing two metal ions. In the case of mono-zinc enzymes, Asp90 hydrogen bonds to the nucleophilic water molecule. The latter is the case for the enzyme BLII. In order to probe the functional and structural role of Asp90 in B.cereus BLII, we have replaced it by Asn, Gln, Glu, His, Ser and Ala through site-directed mutagenesis. The mutants exhibited impaired catalytic efficiencies towards different substrates. Spectroscopic studies on Co(II)-substituted forms of these mutants may help on dissecting the structural effect of these mutations in the active site. These results highlight the role of Asp90 in the catalytic mechanism of B.cereus BLII, that may be extended to other members of this family of enzymes.