Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Department of Bioscience and Biotechnology, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan.
Biochem Biophys Res Commun. 2021 Jan 1;534:286-291. doi: 10.1016/j.bbrc.2020.11.093. Epub 2020 Dec 4.
γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site.
γ-谷氨酰转肽酶（GGT）是一种广泛存在的酶，能够催化 γ-谷氨酰化合物的 γ-谷氨酰键的水解以及其 γ-谷氨酰部分向受体底物的转移。假单胞菌 GGT（PnGGT）用于茶氨酸的工业合成，因此确定水解和转移反应的结构基础并鉴定 PnGGT 的受体部位对于提高茶氨酸合成的效率非常重要。我们之前对 PnGGT 的结构研究表明，三个氨基酸残基色氨酸 385、苯丙氨酸 417 和色氨酸 525 之间的关键相互作用将 PnGGT 与其他 GGT 区分开来。在这里，我们基于定点突变和结构分析报告了色氨酸 525 在 PnGGT 中的作用。产生了七个色氨酸 525 的突变变体（W525F、W525V、W525A、W525G、W525S、W525D 和 W525K），通过非芳香族残基取代色氨酸 525 导致水解活性显著降低。所有色氨酸 525 突变体对羟胺的转移活性显著增加，而对受体底物的偏好几乎没有影响。与谷氨酰胺拮抗剂 6-重氮-5-氧-L-正亮氨酸复合物的 PnGGT 晶体结构表明，色氨酸 525 是限制 PnGGT 活性部位内水分子运动的关键残基。
