Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage β-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-β-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-β-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing β6–α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the −1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

中文翻译：

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糖苷水解酶亚家族 GH5_57 具有高度重新设计的催化界面，可处理复杂的异型β-甘露聚糖

糖苷水解酶家族 5 (GH5) 具有多种底物特异性和作用模式，表现出显着的分子适应性，可应对糖苷和碳水化合物（如纤维素、木葡聚糖、混合键 β-葡聚糖、昆布多糖、（杂）木聚糖）所带来的立体化学复杂性、(杂)甘露聚糖、半乳聚糖、壳聚糖、N-聚糖、芦丁和橙皮苷。GH5 被划分为亚家族，其中许多具有更高的功能特异性，其中一些迄今为止尚未被表征，而另一些则尚未随着对序列/分类多样性的探索而被发现。在这项工作中，通过描述从水豚肠道微生物群中回收的未培养的拟杆菌属细菌的内切-β-甘露聚糖酶 (CapGH5_57) 发现了 GH5_57 亚科，从而扩展了当前的 GH5 亚科库存。生化表征表明CapGH5_57对葡甘聚糖具有活性，释放出聚合度为2至6的寡糖，表明它是一种内切-β-甘露聚糖酶。与 GH5 甘露聚糖酶相比，使用单波长反常衍射解决的晶体结构揭示了大规模重新设计的催化界面。CapGH5_57 中不存在典型的芳族平台和 GH5_7 甘露聚糖酶正亚位点区域中特征性的含 α-螺旋的 β6-α6 环，从而产生可能有利于支链底物结合的大而开放的催化界面。支持这一点的是，CapGH5_57 含有与切割位点相邻并垂直的色氨酸残基，表明在 -1 糖基部分具有取代基的底物的锚定位点。综合起来，这些结果表明，尽管 CapGH5_57 对葡甘露聚糖具有内切活性，但它可能具有一种新型取代的异甘露聚糖作为其天然底物。这项工作通过揭示一种新的催化界面来识别复杂的异甘露聚糖，从而证明了关于这一大型和多特异性 GH 家族的机制和功能多样性的发现仍有很大潜力，这导致了 GH5_57 亚家族的建立。