Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Japan.
Biochem Biophys Res Commun. 2012 Jan 20;417(3):951-5. doi: 10.1016/j.bbrc.2011.12.033. Epub 2011 Dec 16.
Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using L-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate.
最近，我们解析了来自链霉菌 X-119-6 的 L-谷氨酸氧化酶（LGOX）的晶体结构（PDB 代码：2E1M），其底物特异性对 L-谷氨酸非常严格。通过使用 L-谷氨酸和 LGOX 的结构进行对接模拟，我们选择了三个残基，Arg305、His312 和 Trp564，作为与识别 L-谷氨酸相关的残基的候选者。用丙氨酸取代选定的残基后，LGOX 对 L-谷氨酸的活性显著降低。然而，Arg305 被取代为 Ala 的酶对各种 L-氨基酸表现出催化活性。为了研究 Arg305 在底物特异性中的作用，我们构建了 LGOX 的 Arg305 变体。在所有突变体中，LGOX 的底物特异性因突变而明显改变。动力学和活性对 pH 值的依赖性的结果表明，LGOX 的 Arg305 与酶和底物侧链的相互作用有关。
