Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus.
Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus.
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DOI：
10.1042/bj20121541
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发表时间：
2013-04-01
期刊：
影响因子：
--
通讯作者：
Hamilton CJ
中科院分区：
文献类型：
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作者：
Roberts AA;Sharma SV;Strankman AW;Duran SR;Rawat M;Hamilton CJ
FosB是一种二价金属依赖性巯基-S-转移酶，与许多致病性革兰氏阳性菌的磷霉素耐药性有关。在本文中，我们描述了详细的动力学研究FosB从金黄色葡萄球菌（SaFosB），证实了bacillithiol（BSH）是其首选的生理巯基底物。SaFosB是第一个被鉴定的一类新的酶（杆菌锂-S-转移酶），与谷胱甘肽转移酶不同，它分布在许多低G + C革兰氏阳性细菌中，这些细菌使用BSH而不是谷胱甘肽作为其主要的低分子量硫醇。BSH和磷霉素的Km值分别为4.2和17.8 mM。底物特异性分析表明，巯基和氨基的BSH的活性是必不可少的，而苹果酸是重要的SaFosB的识别和催化效率。金属活性测定表明，Mn 2+和Mg 2+可能是生理条件下的相关辅因子。BSH的丝氨酸类似物（BOH）是SaFosB相对于BSH的有效竞争性抑制剂，但相对于磷霉素无竞争性。结合反应产物（BS-磷霉素）的NMR表征，这表明SaFosB催化的反应途径涉及强制有序的结合机制，首先磷霉素结合，然后是BSH，然后BSH攻击磷霉素环氧化物的空间位阻更大的C-1碳。阻断S.金黄色葡萄球菌增加对磷霉素的敏感性。总之，这些结果表明SaFosB是一种二价金属依赖性的杆菌锂硫-S-转移酶，其赋予S.金黄色。
FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G + C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The Km values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn2+ and Mg2+ are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS– fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.