A model for glutathione binding and activation in the fosfomycin resistance protein, FosA.
A model for glutathione binding and activation in the fosfomycin resistance protein, FosA.
复制标题
磷霉素抗性蛋白 FosA 中谷胱甘肽结合和激活的模型。
DOI：
10.1016/j.abb.2007.04.035
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发表时间：
2007
影响因子：
3.9
通讯作者：
Armstrong,RichardN
中科院分区：
文献类型：
--
作者：
Rigsby,RachelE;Brown,DanielW;Dawson,Eric;Lybrand,TerryP;Armstrong,RichardN
来自铜绿假单胞菌的基因组编码的磷霉素抗性蛋白（FosAPA）利用Mn（II）和K+催化谷胱甘肽（GSH）添加到抗生素的C1中，使其失活。虽然这种蛋白质的结构和动力学特征相对于底物，磷霉素，问题仍然存在关于酶如何结合巯基底物，谷胱甘肽。计算研究揭示了FosAPA中一个潜在的GSH结合位点，该位点涉及与蛋白质侧链的六个静电或氢键相互作用以及六个有助于货车范德华相互作用的额外残基。一个战略性放置的酪氨酸残基，Y39，似乎是参与在催化过程中的GSH的电离。Y39F突变体的催化活性降低了13倍（kcat=14±2s−1），这表明它在GSH的电离中起作用。涉及离子或氢键相互作用的其他五个残基（W34、Q36、S50、K90和R93）的突变导致酶具有降低的催化效率、对GSH的亲和力或两者。还发现突变酶在大肠杆菌的生物学背景下是不太有效的抗性蛋白。更保守的W34 H突变体具有类似天然的催化效率，这表明咪唑NH基团可以取代对GSH结合重要的W34的吲哚基团。在没有与巯基底物的共晶结构数据的情况下，这些结果为GSH在催化中的作用提供了重要的见解。
The genomically encoded fosfomycin resistance protein from Pseudomonas aeruginosa (FosAPA) utilizes Mn(II) and K+to catalyze the addition of glutathione (GSH) to C1 of the antibiotic rendering it inactive. Although this protein has been structurally and kinetically characterized with respect to the substrate, fosfomycin, questions remain regarding how the enzyme binds the thiol substrate, GSH. Computational studies have revealed a potential GSH binding site in FosAPAthat involves six electrostatic or hydrogen-bonding interactions with protein side-chains as well as six additional residues that contribute van der Waals interactions. A strategically placed tyrosine residue, Y39, appears to be involved in the ionization of GSH during catalysis. The Y39F mutant exhibits a 13-fold reduction of catalytic activity (kcat=14±2s−1), suggesting a role in the ionization of GSH. Mutation of five other residues (W34, Q36, S50, K90, and R93) implicated in ionic of hydrogen-bonding interactions resulted in enzymes with reduced catalytic efficiency, affinity for GSH, or both. The mutant enzymes were also found to be less effective resistant proteins in the biological context of Escherichia coli. The more conservative W34H mutant has native-like catalytic efficiency suggesting that the imidazole NH group can replace the indole group of W34 that is important for GSH binding. In the absence of co-crystal structural data with the thiol substrate, these results provide important insights into the role of GSH in catalysis.