Engineered Mononuclear Variants in Bacillus cereus Metallo-β-lactamase BcII Are Inactive

• Published in: Biochemistry (Easton) 2008-08, Vol.47 (33), p.8590-8599
• Main Authors: Abriata, Luciano A, González, Lisandro J, Llarrull, Leticia I, Tomatis, Pablo E, Myers, William K, Costello, Alison L, Tierney, David L, Vila, Alejandro J
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; ANTIBIOTICS; BACILLUS CEREUS; Bacillus cereus - enzymology; BACTERIA; BASIC BIOLOGICAL SCIENCES; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; Binding Sites; DESIGN; ENZYMES; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; Models, Molecular; MUTANTS; Mutation; national synchrotron light source; Protein Conformation; Protein Engineering; RESIDUES; ZINC
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi8006912

Metallo-β-lactamases (MβLs) are zinc enzymes able to hydrolyze almost all β-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MβLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-β-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MβL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.