Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis

Simm, Alan M., Higgins, Catherine S., Carenbauer, Anne L., Crowder, Michael W., Bateson, John H., Bennett, Peter M., Clarke, Anthony R., Halford, Stephen E. and Walsh, Timothy Rutland ORCID: https://orcid.org/0000-0003-4315-4096 2002. Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis. Journal of Biological Chemistry 277 (27) , pp. 24744-24752. 10.1074/jbc.M201524200

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Abstract

The L1 metallo-?-lactamase fromStenotrophomonas 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 higherK 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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Medicine Systems Immunity Research Institute (SIURI)
ISSN:	1083-351X
Last Modified:	17 Oct 2022 08:30
URI:	https://orca.cardiff.ac.uk/id/eprint/307

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