Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase.
Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase.
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DOI：
10.1021/bi7017943
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发表时间：
2008-01
期刊：
影响因子：
2.9
通讯作者：
O. Quaye;G. Lountos;F. Fan;A. Orville;G. Gadda
中科院分区：
文献类型：
--
作者：
O. Quaye;G. Lountos;F. Fan;A. Orville;G. Gadda
胆碱氧化酶催化胆碱氧化为甘氨酸甜菜碱，甘氨酸甜菜碱是一种相容的溶质，在病原细菌和植物中积累，因此它们可以承受渗透和温度胁迫。胆碱氧化酶的晶体结构进行了测定，并细化到1.86 A的分辨率与数据收集在100 K，使用同步辐射X射线辐射。该结构揭示了His 99 Nepsilon 2和FAD C8 M原子之间的共价键，以及邻近黄素表面的123 A3溶剂排除空腔。一个假设的模型胆碱停靠到腔表明，几个芳香族残基和Glu 312可以定向的阳离子底物有效的催化。Glu 312上负电荷的作用通过工程化变体酶来研究，其中Glu 312被丙氨酸、谷氨酰胺或天冬氨酸替代。Glu 312 Ala酶无活性。Glu 312 Gln酶对胆碱的Kd值比野生型酶大至少500倍。Glu 312 Asp酶的kcat/KO 2值与野生型酶相似，但kcat和kcat/Km值分别比野生型酶低230倍和35倍。这些数据与残基312上的负电荷的空间位置一致，这对于醇底物的氧化是重要的。溶剂粘度和底物动力学同位素效应表明在Glu 312 Asp酶的氢化物转移反应之前存在内部平衡。总之，晶体学和机械数据表明，Glu 312是重要的结合和定位的胆碱氧化酶的活性位点的基板。
Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.