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首页> 外文期刊>Biochemistry >Structural and Biochemical Insights into the Mechanism of Fosfomycin Phosphorylation by Fosfomycin Resistance Kinase FomA
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Structural and Biochemical Insights into the Mechanism of Fosfomycin Phosphorylation by Fosfomycin Resistance Kinase FomA

机译：磷霉素抗性激酶FomA对磷霉素磷酸化机理的结构和生化研究。

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摘要

We present here the crystal structures of fosfomycin resistance protein (FomA) complexed with MgATP, with ATP and fosfomycin, with MgADP and fosfomycin vanadate, with MgADP and the product of the enzymatic reaction, fosfomycin monophosphate, and with ADP at 1.87, 1.58, 1.85, 1.57, and 1.85 ? resolution, respectively. Structures of these complexes that approximate different reaction steps allowed us to distinguish the catalytically active conformation of ATP and to reconstruct the model of the MgATPfosfomycin complex. According to the model, the triphosphate tail of the nucleotide is aligned toward the phosphonate moiety of fosfomycin, in contast to the previously published MgAMPPNP complex, with the attacking fosfomycin oxygen positioned 4 A from the γ-phosphorus of ATP. Site-directed mutagenesis studies and comparison of these structures with that of homologous N-acetyl-L-glutamate and isopentenyl phosphate kinases allowed us to propose a model of phosphorylation of fosfomycin by FomA enzyme. A Mg cation ligates all three phosphate groups of ATP and together with positively charged K216, K9, K18, and H58 participates in the dissipation of negative charge during phosphoryl transfer, indicating that the transferred phosphate group is highly negatively charged, which would be expected for an associative mechanism. K216 polarizes the γ-phosphoryl group of ATP. K9, K18, and H58 participate in stabilization of the transition state. D150 and D208 play organizational roles in catalysis.S148, S149, and T210 participate in fosfomycin binding, with T210 being crucial for catalysis. Hence, it appears that as in the homologous enzymes, FomA-catalyzed phosphoryl transfer takes place by an in-line predominantly associative mechanism.
机译：我们在这里介绍了磷霉素抗性蛋白（FomA）的晶体结构，与MgATP，ATP和磷霉素，MgADP和磷霉素钒酸盐，MgADP以及酶促反应产物磷磷霉素和ADP的复合结构分别为1.87、1.58、1.85 ，1.57和1.85？分辨率分别。这些复合物的结构接近不同的反应步骤，使我们能够区分ATP的催化活性构象，并重建MgATP磷霉素复合物的模型。根据该模型，与先前公开的MgAMPPNP复合物相比，核苷酸的三磷酸尾与福霉素的膦酸酯部分对齐，而攻击磷则位于距ATPγ-磷4 A的位置。定点诱变研究以及与同源N-乙酰-L-谷氨酸和异戊烯基磷酸激酶的这些结构的比较使我们能够提出一种由FomA酶磷酸化磷霉素的模型。 Mg阳离子连接ATP的所有三个磷酸基团，并且带正电的K216，K9，K18和H58参与磷酰基转移过程中的负电荷耗散，表明转移的磷酸基团高度带负电，这对于关联机制。 K216极化ATP的γ-磷酰基。 K9，K18和H58参与过渡状态的稳定化。 D150和D208在催化中起组织作用.S148，S149和T210参与磷霉素的结合，其中T210对催化至关重要。因此，似乎在同源酶中，FomA催化的磷酸基转移是通过在线主要缔合机制发生的。

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