Vardakou Maria, Dumon Claire, Murray James W, Christakopoulos Paul, Weiner David P, Juge Nathalie, Lewis Richard J, Gilbert Harry J, Flint James E
Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
J Mol Biol. 2008 Feb 1;375(5):1293-305. doi: 10.1016/j.jmb.2007.11.007. Epub 2007 Nov 12.
Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential -3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting beta strands 11 and 12, as suggested previously, but is highly complex.
内切-β-1,4-木聚糖酶（木聚糖酶）可水解木聚糖主链中的β-1,4糖苷键。尽管糖苷水解酶家族11（GH11）中的木聚糖酶已得到广泛研究，但仍有几个问题尚未解决。因此，这些酶水解修饰木聚糖的机制尚不清楚，该家族内催化活性变化的结构基础也未知。此外，小麦蛋白XIP-I对真菌GH11酶抑制作用存在差异的机制仍不明确。为解决这些问题，我们报道了梨形新丽鞭毛虫木聚糖酶NpXyn11A的晶体结构和生化特性，该酶具有异常高的催化活性，是少数不受XIP-I抑制的真菌GH11蛋白之一。尽管无法确定NpXyn11A与底物结合的复合物结构，但我们通过解析另一种GH11木聚糖酶EnXyn11A（由环境DNA样本编码）与阿魏酸-1,5-阿拉伯呋喃糖-α-1,3-木三糖（FAX(3)）结合的晶体结构，得以研究GH11酶如何水解修饰底物。EnXyn11A-FAX(3)复合物的晶体结构表明，-3和+2亚位点处主链木糖O2和O3基团的溶剂暴露允许α-1,2-连接的4-甲基-D-葡萄糖醛酸和L-阿拉伯呋喃糖侧链的容纳。此外，阿魏酸化阿拉伯呋喃糖侧链在+2亚位点形成氢键和疏水相互作用，表明这种修饰可能是该糖苷配基亚位点的特异性决定因素。NpXyn11A的结构揭示了在动力学上具有重要意义的潜在-3和+3亚位点。与其他GH11木聚糖酶相比，NpXyn11A扩展的底物结合裂隙可能解释了梨形新丽鞭毛虫酶为何具有异常高的催化活性。最后，NpXyn11A的晶体结构表明，该酶对XIP-I的抗性并非如先前所述仅归因于β链11和12之间环中的插入，而是高度复杂的。
