Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis.
The L1 metallo-beta-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 beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-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 beta-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 beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.
Simm
Alan M
AM
Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. A.M.Simm@bristol.ac.uk
Higgins
Catherine S
CS
Carenbauer
Anne L
AL
Crowder
Michael W
MW
Bateson
John H
JH
Bennett
Peter M
PM
Clarke
Anthony R
AR
Halford
Stephen E
SE
Walsh
Timothy R
TR
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics*; D001483:Base Sequence; D001711:Biotransformation; D018360:Crystallography, X-Ray; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020615:Stenotrophomonas maltophilia / Q000201:enzymology*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; C088032:beta-lactamase L1; D001618:beta-Lactamases