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J. Biol. Chem., Vol. 277, Issue 27, 24744-24752, July 5, 2002

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Characterization of Monomeric L1 Metallo--lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis^*

Alan M. Simm^§, Catherine S. Higgins, Anne L. Carenbauer^¶, Michael W. Crowder^¶, John H. Bateson, Peter M. Bennett, Anthony R. Clarke^**, Stephen E. Halford^**, and Timothy R. Walsh

From the  Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom, the ^¶ Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, the  Department of Medicinal Chemistry, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AW, United Kingdom, and the ^** Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom

The L1 metallo--lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in -lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 -lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of -lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K_m values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K_m values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for -lactam hydrolysis, it is clearly important to its activity and substrate specificity.

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