Structural insights reveal the second base catalyst of isomaltose glucohydrolase
Structural insights reveal the second base catalyst of isomaltose glucohydrolase
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结构见解揭示了异麦芽糖葡萄糖水解酶的第二碱催化剂
DOI：
10.1111/febs.16237
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Kimura Atsuo
中科院分区：
文献类型：
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作者：
Tagami Takayoshi;Chen Minghao;Furunaga Yuta;Kikuchi Asako;Sadahiro Juri;Lang Weeranuch;Okuyama Masayuki;Tanaka Yoshikazu;Iwasaki Tomohito;Yao Min;Kimura Atsuo
糖苷水解酶家族15 （GH15）转化酶含有两个谷氨酸残基，分别作为异麦芽糖葡萄糖水解酶（IGHase）的一般酸催化剂和一般碱催化剂，分别为Glu178和Glu335。一般来说，双催化残基介导的反应呈现出典型的钟形ph -活性曲线。然而，IGHase显示出非典型的非钟形pH - kcat和pH - kcat/ km曲线，理论上更符合三催化残留物相关的pH -活性曲线。我们利用硫原子和催化碱基突变体E335A的异麦芽糖共晶结构，用单波长异常色散法测定了IGHase的晶体结构。虽然检测不到E335A的活性，但在其活性位点口袋中观察到的电子密度不对应于异麦芽糖，而是对应于甘油和β -葡萄糖，冷冻保护剂和水解产物。我们对几种突变酶的结构和生化分析表明，Tyr48作为第二催化碱基催化剂。Y48F突变体表现出与催化酸突变体E178A几乎相同的比活性。Tyr48在所有GH15成员中高度保守，在许多GH15酶中被另一个Tyr残基固定；后者Tyr在IGHase中被Phe290取代。F290Y突变体的pH谱变化为钟形曲线，表明Phe290是区分IGHase Tyr48与其他GH15成员的关键残基。此外，F290Y通过改变Tyr290 - Tyr48 - Glu335之间的氢键网络，加速了葡萄糖与异麦芽糖的缩合。本研究表明，非典型Phe290使IGHase的Tyr48在GH15酶中是独一无二的。
Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two‐catalytic residue‐mediated reaction exhibits a typical bell‐shaped pH–activity curve. However, IGHase is found to display atypical non‐bell‐shaped pH‐kcatand pH‐kcat/Kmprofiles, theoretically better‐fitted to a three‐catalytic residue‐associated pH–activity curve. We determined the crystal structure of IGHase by the single‐wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a β‐glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell‐shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen‐bonding network between Tyr290‐Tyr48‐Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.