title: Overexpression, purification, and characterization of the cloned metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.
abstract: The metallo-beta-lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, with k(cat)/Km values ranging between 0.002 and 5.5 microM(-1) s(-1). These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-beta-lactamases isolated directly from S. maltophilia.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA. crowdemw@muohio.edu
authors.forename: M W
authors.initials: MW
authors.lastname: Crowder
authors.forename: T R
authors.initials: TR
authors.lastname: Walsh
authors.forename: L
authors.initials: L
authors.lastname: Banovic
authors.forename: M
authors.initials: M
authors.lastname: Pettit
authors.forename: J
authors.initials: J
authors.lastname: Spencer
mesh_terms: D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008670:Metals / Q000032:analysis; D008970:Molecular Weight; D010957:Plasmids; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013054:Spectrophotometry, Atomic; D014974:Xanthomonas / Q000201:enzymology*; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D008670:Metals; C088032:beta-lactamase L1; D001618:beta-Lactamases