Thongpoo Preeyanuch, McKee Lauren S, Araújo Ana Catarina, Kongsaeree Prachumporn T, Brumer Harry
Interdisciplinary Graduate Program in Genetic Engineering, Faculty of Graduate School, Kasetsart University, Bangkok 10900, Thailand.
Biochim Biophys Acta. 2013 Mar;1830(3):2739-49. doi: 10.1016/j.bbagen.2012.11.014.
The commercially important glycoside hydrolase family 3 (GH3) beta-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined.
In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 beta-glucosidase from A. niger ASKU28.
In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k(cat) and k(cat)/K(m) values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl beta-D-glucoside and 2,4-dinitrophenyl beta-D-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl beta-glucosides and yielded beta-glucosyl azide as a product.
These results strongly support the assignment of E490 as the acid/base catalyst in a beta-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.
来自黑曲霉的具有商业重要性的糖苷水解酶家族3（GH3）β-葡萄糖苷酶是保留异头构型的酶，其通过典型的双置换糖苷酶机制发挥作用。尽管通过序列比对在所有GH3成员中很容易识别出催化亲核试剂，但该家族中的酸/碱催化剂在系统发育上是可变的，不太容易推测出来。
在本报告中，我们采用三维结构同源建模和定点突变体的详细动力学分析来鉴定来自黑曲霉ASKU28的GH3β-葡萄糖苷酶的催化酸/碱。
与野生型酶和其他突变体相比，E490A变体对天然底物纤维二糖的k(cat)和k(cat)/K(m)值大幅降低（分别降低了67,000倍和61,000倍）。对于人工生色底物对硝基苯基β-D-葡萄糖苷和2,4-二硝基苯基β-D-葡萄糖苷，观察到相应较小的动力学效应，其糖苷配基离去基团对酸催化的依赖性较小，尽管两者的速率决定催化步骤都有变化。pH-速率曲线分析也表明E490是一般酸/碱催化剂。添加叠氮化物作为外源亲核试剂可部分恢复E490A变体与芳基β-葡萄糖苷的活性，并产生β-葡萄糖基叠氮化物作为产物。
这些结果有力地支持了将E490指定为黑曲霉ASKU28的β-葡萄糖苷酶中的酸/碱催化剂，并为一系列相关GH3亚家族成员中同源残基的生物信息学鉴定提供了关键的实验支持。
