Fillgrove Kerry L, Pakhomova Svetlana, Newcomer Marcia E, Armstrong Richard N
Departments of Biochemistry and Chemistry, Vanderbilt University, Nashville, Tennessee 37232-0146, USA.
J Am Chem Soc. 2003 Dec 24;125(51):15730-1. doi: 10.1021/ja039307z.
Microbial resistance to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonic acid, 1] is known to be mediated by thiol transferase enzymes FosA and FosB, which catalyze the addition of glutathione and l-cysteine to C1 of the oxirane, respectively. A probe of the microbial genome database reveals a related group of enzymes (FosX). The genes mlr3345 from Mesorhizobium loti and lmo1702 from Listeria monocytogenes were cloned and the proteins expressed. This heretofore unrecognized group of enzymes is shown to catalyze the Mn(II)-dependent addition of water to C1 of the oxirane. The ability of each enzyme to confer resistance in Escherichia coli is correlated with their catalytic efficiency, such that the M. loti protein confers low resistance while the Listeria enzyme confers very robust resistance. The crystal structure of the FosX from M. loti was solved at a resolution of 1.83 A. The structure reveals an active-site carboxylate (E44) located about 5 A from the expected position of the substrate that appears to be poised to participate in catalysis. Single turnover experiments in H218O and kinetic analysis of the E44G mutant of the FosX enzymes indicate that the carboxylate of E44 acts as a general base in the direct addition of water to 1. The FosX from M. loti also catalyzes the addition of glutathione to the antibiotic. The catalytic promiscuity and low efficiency of the M. loti protein suggest that it may be an intermediate in the evolution of clinically relevant fosfomycin resistance proteins such as the FosX from Listeria monocytogenese.
已知微生物对抗生素磷霉素[（1R，2S）-环氧丙基膦酸，1]的抗性是由硫醇转移酶FosA和FosB介导的，它们分别催化谷胱甘肽和L-半胱氨酸加到环氧乙烷的C1位上。对微生物基因组数据库的探查揭示了一组相关的酶（FosX）。克隆了来自百脉根中生根瘤菌的mlr3345基因和来自单核细胞增生李斯特菌的lmo1702基因，并表达了相应的蛋白质。结果表明，这一此前未被认识的酶组催化环氧乙烷C1位上依赖于Mn（II）的水加成反应。每种酶在大肠杆菌中赋予抗性的能力与其催化效率相关，因此百脉根中的蛋白质赋予低抗性，而李斯特菌中的酶赋予很强的抗性。以1.83 Å的分辨率解析了来自百脉根的FosX的晶体结构。该结构揭示了一个活性位点羧酸盐（E44），其位于距底物预期位置约5 Å处，似乎准备参与催化反应。在H218O中进行的单周转实验以及FosX酶的E44G突变体的动力学分析表明，E44的羧酸盐在水直接加成到1的反应中起通用碱的作用。来自百脉根的FosX还催化谷胱甘肽加到该抗生素上。百脉根中蛋白质的催化多效性和低效率表明，它可能是临床上相关的磷霉素抗性蛋白（如来自单核细胞增生李斯特菌的FosX）进化过程中的一个中间体。
