7-木糖紫杉烷糖基水解酶LXYL-P1-1和LXYL-P1-2活性中心预测及初步的功能分析

Prediction and preliminary functional analysis of the potential active center of the glycoside hydrolases LXYL-P1-1 and LXYL-P1-2

7-木糖紫杉烷糖基水解酶LXYL-P1-1和LXYL-P1-2是克隆自真菌香菇的两个双功能酶(序列一致性97%),具有β-木糖苷酶/β-葡萄糖苷酶双重活性,能特异性地水解移除7-木糖-10-去乙酰紫杉醇等紫杉烷上的木糖基。采用生物信息学方法对两个酶蛋白进行酶活性中心预测,初步确定Asp300和Glu529分别为亲核试剂和一般酸/碱催化剂,而Asn172-Gly173-Arg174和Lys207-His208为底物结合结构域。以LXYL-P1-2为研究对象,以毕赤酵母细胞为表达宿主,应用定点突变技术获得了N172A、G173A、R174A、K207A、H208A、D300N和E529Q突变体,并进行了酶活性分析。结果显示:在分别以PNP-Xyl、PNP-Glc和7-木糖-10-去乙酰紫杉醇为底物时,N172A、G173A、R174A、K207A、D300N和E529Q的β-木糖苷酶与β-葡萄糖苷酶活性大幅度下降甚至完全消失;H208A的β-木糖苷酶活性也显著下降,但仍保持98%的β-葡萄糖苷酶活性。其结果初步验证了对上述两个酶蛋白的活性中心的预测,为进一步揭示7-木糖紫杉烷糖基水解酶结构与功能的关系提供了实验依据。

Glycoside hydrolases of 7-xylosyltaxanes LXYL-PI-1 and LXYL-P1-2 are two bifunctional β-D-xylosidases/ β-D-glucosidases cloned from Lentinula edodes. The two enzymes （identity 97%） could specifically remove the xylosyl residue from 7-xylosyltaxanes such as 7-xylosyl-10-deacetyltaxol （XDT）. Bioinformatics method was applied to predict the active center of the two enzymes and preliminarily determined Asp300 as the catalytic nucleophile, Glu529 as the general acid/base catalyst, Asn172-Gly173-ArgJ74and Lys207-His208 as the two conserved motifs for the substrate binding, respectively. The LXYL-P1-2 mutants, including N172A, G173A, R174A, K207A, H208A, D300N and E529Q, were obtained by means of the site-directed mutagenesis technique, with the yeast Pichia pastoris as the expression host. All the mutants were subjected to the enzyme activity analysis. Results showed that both the β-D-xylosidase and β-D-glucosidase activities of N172A, G173A, R174A, K207A, D300N and E529Q were dramatically decreased or even lost. The β-D-xylosidase activity of H208A was also obviously decreased, but it still kept 98% of the β-D-glucosidase activity. The results preliminarily supported the prediction of the active center and provided an experimental basis for further study of the relationship between the structure and function of the glycoside hydrolases of 7-xylosyltaxanes.