Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

6-22-2007

Publisher

American Society for Biochemistry and Molecular Biology

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Original Item ID

doi: 10.1074/jbc.M700742200

Abstract

Metallo-β-lactamases are zinc-dependent hydrolases that inactivate β-lactam antibiotics, rendering bacteria resistant to them. Asp-120 is fully conserved in all metallo-β-lactamases and is central to catalysis. Several roles have been proposed for Asp-120, but so far there is no agreed consensus. We generated four site-specifically substituted variants of the enzyme BcII from Bacillus cereus as follows: D120N, D120E, D120Q, and D120S. Replacement of Asp-120 by other residues with very different metal ligating capabilities severely impairs the lactamase activity without abolishing metal binding to the mutated site. A kinetic study of these mutants indicates that Asp-120 is not the proton donor, nor does it play an essential role in nucleophilic activation. Spectroscopic and crystallographic analysis of D120S BcII, the least active mutant bearing the weakest metal ligand in the series, reveals that this enzyme is able to accommodate a dinuclear center and that perturbations in the active site are limited to the Zn2 site. It is proposed that the role of Asp-120 is to act as a strong Zn2 ligand, locating this ion optimally for substrate binding, stabilization of the development of a partial negative charge in the β-lactam nitrogen, and protonation of this atom by a zinc-bound water molecule.

Comments

Published version. Journal of Biological Chemistry, Vol. 282, No. 25 (June 22, 2007): 18276-18285. DOI. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Used with permission.

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