Substrate specificity of glycoside hydrolase family 1 β-glucosidase AtBGlu42 from Arabidopsis thaliana and its molecular mechanism
Substrate specificity of glycoside hydrolase family 1 β-glucosidase AtBGlu42 from Arabidopsis thaliana and its molecular mechanism
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DOI：
10.1093/bbb/zbab200
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发表时间：
2022-01-24
影响因子：
1.6
通讯作者：
Mori, Haruhide
中科院分区：
文献类型：
--
作者：
Horikoshi, Shu;Saburi, Wataru;Mori, Haruhide
植物具有许多糖苷水解酶家族1（GH 1）β-葡糖苷酶，其在细胞壁代谢和生物活性物质的活化中发挥生理功能，但大多数仍未被表征。拟南芥中的一种GH 1同工酶AtBGlu 42已被鉴定为利用基因缺陷植物水解东莨菪碱，但没有获得酶性质。AtBGlu 42与水稻中另一种功能酶Os 1BGlu 4的序列相似性表明AtBGlu 42也作用于寡糖。在这里，我们表明，重组AtBGlu 42具有高k（猫）/K-m不仅对东莨菪碱，但也对各种β-葡萄糖苷，纤维寡糖，海带寡糖。在纤维低聚糖中，纤维三糖是最优选的。在1.7埃分辨率下测定的晶体结构表明，Arg 342在亚位点+3处与纤维寡糖产生不利的结合。突变体R342 Y和R342 A显示出对纤维四糖或纤维五糖的最高偏好，在亚位点+3处具有增加的亲和力，表明在该位置处的残基对链长特异性具有重要作用。
Plants possess many glycoside hydrolase family 1 (GH1) beta-glucosidases, which physiologically function in cell wall metabolism and activation of bioactive substances, but most remain uncharacterized. One GH1 isoenzyme AtBGlu42 in Arabidopsis thaliana has been identified to hydrolyze scopolin using the gene deficient plants, but no enzymatic properties were obtained. Its sequence similarity to another functionally characterized enzyme Os1BGlu4 in rice suggests that AtBGlu42 also acts on oligosaccharides. Here, we show that the recombinant AtBGlu42 possesses high k(cat)/K-m not only on scopolin, but also on various beta-glucosides, cellooligosaccharides, and laminarioligosaccharides. Of the cellooligosaccharides, cellotriose was the most preferred. The crystal structure, determined at 1.7 angstrom resolution, suggests that Arg342 gives unfavorable binding to cellooligosaccharides at subsite +3. The mutants R342Y and R342A showed the highest preference on cellotetraose or cellopentaose with increased affinities at subsite +3, indicating that the residues at this position have an important role for chain length specificity.