Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14
Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14
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DOI：
10.1074/jbc.m115.691659
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发表时间：
2016-01-15
影响因子：
4.8
通讯作者：
Brumer, Harry
中科院分区：
文献类型：
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作者：
McGregor, Nicholas;Morar, Mariya;Brumer, Harry
最近将糖苷水解酶家族5(GH5)成员划分为亚家族，通过系统发育分析加强了底物专一性的预测。然而，少数具有良好特征的成员是目前理解在单个GH5亚家族中观察到的不同特异性的分子基础的一个限制。GH5亚家族4(GH5_4)是最大的家族之一，具有已知的活性，包括(羧甲基)纤维素酶、混合连接内切葡聚糖酶和内切木糖葡聚糖酶。通过详细的结构-功能分析，我们重新审视了从共生的瘤胃拟杆菌Prevoella bryantii B(1)4中分离出的经典的GH5_4羧甲基纤维素酶PbGH5A(也称为Orf4、羧甲基纤维素酶和Cel5A)的性质。我们证明了PbGH5A实际上是相对较差的底物，而PbGH5A对植物贮藏和细胞壁多糖、混合连接β-葡聚糖表现出明显的初级专一性。对植物细胞壁多糖木糖葡聚糖也有明显的活性。PbGH5A在脱脂形式和与(木糖)低聚糖的络合物中的晶体结构的测定，以及活性部位亲和标记，以及使用各种明确的低聚糖底物的详细的动力学分析，揭示了多糖底物专一性的结构决定因素。特别是，这一分析突出了在GH5_4中产生优势混合连锁内切葡聚糖酶活性的PbGH5A活性位点基序。然而，对GH5_4成员的详细系统发育分析并没有描绘出共享这些序列基序的特定酶类；相反，系统发育由细菌分类主导。然而，我们的结果为未来对(元)基因组的生物信息学分析提供了关键的酶功能和结构参考数据，以阐明复杂肠道生态系统的生物学。
The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl) cellulases, mixedlinkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B(1)4. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixedlinkage beta-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo) glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis a vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta) genomes to elucidate the biology of complex gut ecosystems.