Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens
Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens
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硝基还原假单胞菌 γ-谷氨酰转肽酶色氨酸 525 作用的诱变和基于结构的分析
DOI：
10.1016/j.bbrc.2020.11.093
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发表时间：
2021
影响因子：
3.1
通讯作者：
Hibi Takao
中科院分区：
文献类型：
--
作者：
Sano Chiharu;Itoh Takafumi;Phumsombat Putthapong;Hayashi Junji;Wakayama Mamoru;Hibi Takao
γ-谷氨酰转肽酶（GGT）是一种普遍存在的酶，催化γ-谷氨酰化合物的γ-谷氨酰键的水解并将其γ-谷氨酰部分转移到受体底物上。硝基还原假单胞菌GGT（PnGGT）用于茶氨酸的工业合成，因此，确定PnGGT水解和转移反应的结构基础以及确定PnGGT的受体位点对于提高茶氨酸的效率具有重要意义合成.我们以前对PnGGT的结构研究表明，Trp 385、Phe 417和Trp 525这三个氨基酸残基之间的关键相互作用将PnGGT与其他GGT区分开来。在这里，我们报告的作用Trp 525在PnGGT的基础上的定点突变和结构分析。产生了Trp 525的七种突变变体（W525 F、W525 V、W525 A、W525 G、W525 S、W525 D和W525 K），其中Trp 525被非芳香族残基取代，导致水解活性显著降低。所有Trp 525突变体表现出显着增加转移活性对羟胺几乎没有任何影响受体底物的偏好。与谷氨酰胺拮抗剂6-重氮-5-氧代-1-正亮氨酸复合的PnGGT的晶体结构显示Trp 525是限制PnGGT活性位点内水分子运动的关键残基。
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.PseudomonasnitroreducensGGT (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.