Crowder M W, Walsh T R, Banovic L, Pettit M, Spencer J
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Antimicrob Agents Chemother. 1998 Apr;42(4):921-6. doi: 10.1128/AAC.42.4.921.
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.
嗜麦芽窄食单胞菌的金属β-内酰胺酶L1通过光谱和生化技术进行克隆、过表达及表征。金属分析结果表明，克隆的酶每个单体含有2摩尔紧密结合的Zn(II)。凝胶过滤色谱显示，克隆的酶以紧密结合的四聚体形式存在，分子量约为115 kDa，基质辅助激光解吸电离和飞行时间质谱表明单体分子量为28.8 kDa。对多种青霉素和头孢菌素抗生素进行的稳态动力学研究表明，L1能有效水解所有测试化合物，k(cat)/Km值在0.002至5.5 μM-1 s-1之间。重组酶的这些特性与先前直接从嗜麦芽窄食单胞菌中分离得到的金属β-内酰胺酶的特性形成对比。
