Analysis of the interacting network beneath the metallo-beta-lactamases’ active site

GONZÁLEZ LJ; VILA AJ

Salta

Congreso; 3rd Latin American Protein Society Meeting (LAPS); 2010

Latin American Protein Society

Metallo-beta-lactamases (MbLs) are bacterial zinc-dependent enzymes able to hydrolyze a broad range of beta-lactam antibiotics. Their active sites consist of a highly conserved Zn(II)-binding motif. According to sequence homology MbLs are divided into three classes: B1, B2 and B3. B1 MbLs are broad-range bimetallic enzymes that share a conserved network of H bonding interactions underneath their active sites. This network, comprising conserved residues D84 and R121, is involved in the fine tuning of active site structure. P. aeruginosa SPM-1 is unique among B1 enzymes in that it possess a disrupted network beneath its active site, due to the presence of substitutions D84S and R121G1.   In the present work we attempt to understand the role of the conserved interacting network beneath B1 active sites. In this sense, we randomized codons in SPM-1 gene corresponding to positions 84, 121 and both at the same time. Mutants capable of conferring some degree of resistance to P. aeruginosa PAO against different types of beta-lactam antibiotics were selected. We found that both positions can accommodate residues different from the wild type ones. As judged by minimal inhibitory concentration values, single mutants G121A, D84N and D84G resulted in efficient enzymes, and double mutants showed even more efficiency than wild type SPM-1 against some beta-lactam antibiotics. In addition, residue variations at position 121 markedly influenced substrate specificity. This data will contribute to understanding the role of conserved second-shell interacting networks surrounding the active sites of B1 MbLs. References: 1. Murphy TA, Catto LE, Halford SE, Hadfield AT, Minor W, Walsh TR, Spencer J. (2006). J Mol Biol. 357(3):890-903. Acknowledgments: LJG is recipient of a doctoral fellowship from CONICET. AJV is a Staff member from CONICET and International Research Scholar of the Howard Hughes Medical Institute. This work has been supported by grants from ANPCyT and HHMI to AJV.