Glycoside hydrolase (GH) 30 family xylanases are enzymes of biotechnological interest due to their capacity to degrade recalcitrant hemicelluloses, such as glucuronoxylan (GX). This study focuses on a subfamily 7 GH30, TtXyn30A from Thermothelomyces thermophilus, which acts on GX in an “endo” and “exo” mode, releasing methyl‐glucuronic acid branched xylooligosaccharides (XOs) and xylobiose, respectively. The crystal structure of inactive TtXyn30A in complex with 23‐(4‐O‐methyl‐α‐D‐glucuronosyl)‐xylotriose (UXX), along with biochemical analyses, corroborate the implication of E233, previously identified as alternative catalytic residue, in the hydrolysis of decorated xylan. At the −1 subsite, the xylose adopts a distorted conformation, indicative of the Michaelis complex of TtXyn30AEE with UXX trapped in the semi‐functional active site. The most significant structural rearrangements upon substrate binding are observed at residues W127 and E233. The structures with neutral XOs, representing the “exo” function, clearly show the nonspecific binding at aglycon subsites, contrary to glycon sites, where the xylose molecules are accommodated via multiple interactions. Last, an unproductive ligand binding site is found at the interface between the catalytic and the secondary β‐domain which is present in all GH30 enzymes. These findings improve current understanding of the mechanism of bifunctional GH30s, with potential applications in the field of enzyme engineering.

中文翻译：

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葡萄糖醛酸木聚糖特异性双功能糖苷水解酶 30 木聚糖酶的结构和分子见解

糖苷水解酶 (GH) 30 家族木聚糖酶是具有生物技术意义的酶，因为它们能够降解顽固的半纤维素，例如葡糖醛酸木聚糖 (GX)。本研究重点关注 GH30 亚科 7，ttXyn30A 来自嗜热嗜热酵母，以“内切”和“外切”模式作用于 GX，分别释放甲基葡萄糖醛酸支链低聚木糖 (XO) 和木二糖。非活性晶体结构ttXyn30A与2的复合物3‐(4‐氧-甲基-α-D‐葡萄糖醛酸基)‐木三糖 (UXX) 以及生化分析证实了 E233（之前被确定为替代催化残基）在修饰木聚糖水解中的作用。在 -1 子位点，木糖采用扭曲的构象，表明米氏复合体ttXyn30AEE 与 UXX 被困在半功能活性位点中。在残基 W127 和 E233 处观察到底物结合后最显着的结构重排。具有中性 XO 的结构代表“exo”功能，清楚地显示了苷元亚位点的非特异性结合，这与木糖分子通过多重相互作用容纳的糖基位点相反。最后，在催化结构域和次级 β 结构域之间的界面处发现了一个非生产性配体结合位点，该结构域存在于所有 GH30 酶中。这些发现提高了目前对双功能 GH30 机制的理解，在酶工程领域具有潜在的应用前景。