Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, British Columbia V8W 3P6, Canada.
J Biol Chem. 2012 Apr 20;287(17):13985-95. doi: 10.1074/jbc.M112.345645. Epub 2012 Mar 5.
Agars are abundant polysaccharides from marine red algae, and their chemical structure consists of alternating D-galactose and 3,6-anhydro-L-galactose residues, the latter of which are presumed to make the polymer recalcitrant to degradation by most terrestrial bacteria. Here we study a family 117 glycoside hydrolase (BpGH117) encoded within a recently discovered locus from the human gut bacterium Bacteroides plebeius. Consistent with this locus being involved in agarocolloid degradation, we show that BpGH117 is an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase that removes the 3,6-anhydrogalactose from the non-reducing end of neoagaro-oligosaccharides. A Michaelis complex of BpGH117 with neoagarobiose reveals the distortion of the constrained 3,6-anhydro-L-galactose into a conformation that favors catalysis. Furthermore, this complex, supported by analysis of site-directed mutants, provides evidence for an organization of the active site and positioning of the catalytic residues that are consistent with an inverting mechanism of catalysis and suggests that a histidine residue acts as the general acid. This latter feature differs from the vast majority of glycoside hydrolases, which use a carboxylic acid, highlighting the alternative strategies that enzymes may utilize in catalyzing the cleavage of glycosidic bonds.
琼脂是一种丰富的海洋红藻多糖，其化学结构由交替的 D-半乳糖和 3,6-脱水-L-半乳糖残基组成，后者被认为使聚合物对大多数陆地细菌的降解具有抗性。在这里，我们研究了一种来自人类肠道细菌拟杆菌的最近发现的基因座中编码的家族 117 糖苷水解酶（BpGH117）。与该基因座参与琼脂胶降解一致，我们表明 BpGH117 是一种外切 3,6-脱水-α-(1,3)-L-半乳糖酶，它从 neoagaro-寡糖的非还原端去除 3,6-脱水半乳糖。BpGH117 与 neoagarobiose 的迈克尔is 复合物揭示了受约束的 3,6-脱水-L-半乳糖的扭曲，使其有利于催化。此外，该复合物，通过定点突变体的分析提供了证据，证明了活性位点的组织和催化残基的定位，这与催化的反转机制一致，并表明组氨酸残基作为广义酸。后一特征与绝大多数糖苷水解酶不同，后者使用羧酸，突出了酶在催化糖苷键断裂时可能利用的替代策略。
