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J. Biol. Chem., Vol. 282, Issue 25, 18286-18293, June 22, 2007

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The Metallo--lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site^*

Jorgelina Morán-Barrio¹², Javier M. González¹³, María Natalia Lisa, Alison L. Costello, Matteo Dal Peraro^¶, Paolo Carloni^||4, Brian Bennett^**5, David L. Tierney, Adriana S. Limansky⁶, Alejandro M. Viale⁷, and Alejandro J. Vila⁸

From the Departamento de Química Biológica and Departamento de Microbiología, Instituto de Biología Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina, the Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, the ^¶Center for Molecular Modeling, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the ^||International School for Advanced Studies, Via Beirut 2-4, 34100 Trieste, Italy, and the ^**National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-0509

Metallo--lactamases (MLs) are zinc-dependent enzymes able to hydrolyze and inactivate most -lactam antibiotics. The large diversity of active site structures and metal content among MLs from different sources has limited the design of a pan-ML inhibitor. Here we report the biochemical and biophysical characterization of a novel ML, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp¹²⁰, His¹²¹, His²⁶³, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MLs. Contrasting all other related MLs, GOB-18 is fully active against a broad range of -lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MLs.

Received for publication, January 17, 2007 , and in revised form, March 27, 2007.

^* This work was supported in part by grants from the Agencia Nacional de Promoción Cientifica y Tecnológica (ANPCyT) (to A. J. V. and A. M. V.), the Howard Hughes Medical Institute (HHMI) (to A. J. V.), and from the Biomedical Research Infrastructure Networks/IDeA Networks of Biomedical Research Excellence Program of the National Center for Research Resources (RR16480) (to D. L. T.). M. N. L.'s work was supported by a grant from HHMI (to A. J. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures, Refs. 1–16, Figs. S1–S3, and Table S1.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) DQ004496.

¹ These two authors contributed equally to this work.

² Former fellow of the ANPCyT and a recipient of a doctoral fellowship from Consejo Nacional de Investigaciones Cienificas y Tecnicas (CONICET).

³ Fellow of the ANPCyT.

⁴ Supported by the Istituto Nazionale per la Fisica della Materia and the Ministero dell Universita e della Ricerca Scientifica e Tecnologia-Cofinanziamento.

⁵ Supported by National Institutes of Health Grants AI056231 and RR001980.

⁶ Supported by the Comisión Nacional de Programas de Investigación Sanitaria (Becas Carrillo-Oñativia 2004–2005) and the Departamento de Salud Pública, Municipalidad de Rosario.

⁷ Staff member from CONICET.

⁸ Staff member from CONICET. An International Research Scholar of the HHMI. To whom correspondence should be addressed. Tel.: 54-341-4351235 (ext. 108); Fax: 54-341-4390465; E-mail: vila{at}ibr.gov.ar.

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