Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava, Slovakia.
Carbohydr Res. 2013 Feb 15;367:48-57. doi: 10.1016/j.carres.2012.11.022. Epub 2012 Dec 3.
Among the glycoside hydrolases (GHs) classified within the Carbohydrate-Active enZymes (CAZy) server, the α-amylase family GH13 belongs to the largest GH families. It has been divided into the official 36 subfamilies by the CAZy curators. Originally the subfamilies of oligo-1,6-glucosidase and neopullulanase were defined using the sequence of the fifth conserved sequence region (CSR) as a selection marker. It is localized outside the catalytic α-amylase (β/α)(8)-barrel in the domain B, that is, in a longer loop connecting the strand β3 with the helix α3 of the barrel. It is sequentially positioned 26-28 residues in front of the invariant aspartic acid residue in the β4-strand acting as the GH13 catalytic nucleophile. The CSR V is characteristic as QpDln and MpKln for the former and latter subfamilies, respectively. A group of intermediate sequences possessing the CSR V as a mix of the two above-mentioned subfamilies, that is, MpDln, was also proposed previously. The present bioinformatics analysis was done in an effort to reveal as many as possible GH13 members of this intermediary group, currently classified as the subfamily GH13_36, and to discuss their evolutionary relationships to known GH13 specificities as well as with regard to their taxonomic origin. Using the BLAST tool with the sequence of the α-amylase from Halothermothrix orenii AmyA exhibiting the intermediary features, 152 GH13 enzymes, and hypothetical proteins were retrieved covering defined specificities (GH13 subfamilies 4, 16, 17, 18, 20, 21, 23, 29, 30, 31, 34, and 35) and intermediary enzymes and proteins (GH13_36). In both evolutionary trees-based on the alignment of CSRs and complete sequences-most of the 'intermediary' proteins (i.e., those with MPDLN signature) were positioned in several closely related clusters forming, however, a single GH13_36 large part of the trees. A few novel GH13 subfamilies were proposed as well as the specificity implications were discussed based on the presented in silico analysis. The results may also be helpful in assigning any GH13-like amino acid sequence the subfamily GH13_36 affiliation without additional biochemical characterization.
在碳水化合物活性酶 (CAZy) 服务器中分类的糖苷水解酶 (GHs) 中，α-淀粉酶家族 GH13 属于最大的 GH 家族之一。CAZy 管理员已经将其分为 36 个官方亚家族。最初，寡糖 1,6-葡糖苷酶和新普鲁兰酶的亚家族是使用第五个保守序列区 (CSR) 的序列作为选择标记来定义的。它位于催化 α-淀粉酶 (β/α)(8)-桶的域 B 之外，即连接β3 链和桶的α3 螺旋的较长环中。它位于β4 链中不变天冬氨酸残基的 26-28 个残基之前，该残基作为 GH13 催化亲核体。CSR V 分别是前亚家族 QpDln 和后亚家族 MpKln 的特征。先前还提出了一组具有 CSR V 的中间序列，这些序列是上述两个亚家族的混合物，即 MpDln。本生物信息学分析旨在揭示尽可能多的该中间组的 GH13 成员，目前分类为亚家族 GH13_36，并讨论它们与已知 GH13 特异性以及它们的分类起源的进化关系。使用包含中介特征的 Halothermothrix orenii AmyA 的α-淀粉酶序列的 BLAST 工具，检索了 152 种 GH13 酶和假设蛋白，涵盖了定义的特异性（GH13 亚家族 4、16、17、18、20、21、23、29、30、31、34 和 35）和中介酶和蛋白（GH13_36）。在基于 CSR 和完整序列比对的两个进化树中，大多数“中间”蛋白（即具有 MPDLN 特征的蛋白）位于几个紧密相关的聚类中，然而，在树的大部分中形成了单个 GH13_36。还提出了一些新的 GH13 亚家族，并根据提出的计算机分析讨论了特异性含义。该结果还可能有助于在无需额外生化表征的情况下将任何 GH13 样氨基酸序列分配给 GH13_36 亚家族。
