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.

FIG. 1

SDS-polyacrylamide gel of L1 purification. Lane 1, Novagen perfect protein molecular weight markers; lane 2, boiled cell fraction of BL21(DE3)pLysS containing pUB5832 before induction; lane 3, boiled cell fraction of BL21(DE3)pLysS containing pUB5832 after a 2-h induction with 1 mM IPTG; lane 4, crude protein after French press; lane 5, purified L1. Molecular weights are noted at the left.

FIG. 2

MALDI-TOF mass spectrogram of recombinant L1. The labeled peaks exhibit values of 28,844 m/z for the L1 monomer ([M+H⁺]⁺) and 57,735 m/z for the L1 dimer ([2M+H⁺]⁺). See Materials and Methods for sample conditions. R.I., relative intensity.