Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.
Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.
复制标题
对海栖热袍菌 GH36 α-半乳糖苷酶 (TmGalA) 的生化分析证实了 GH-D 糖苷水解酶家族的机制共性。
作者：
Donald A. Comfort;K. S. Bobrov;D. R. Ivanen;K. Shabalin;James M. Harris;A. Kulminskaya;H. Brumer;R. Kelly
将糖苷水解酶 (GH) 家族组织成族，扩大了成员酶催化机制信息的用途。通过对海栖热袍菌 (TmGalA) 的 GH36 α-半乳糖苷酶进行生化分析，对 GH27 和 GH36 的问题进行了检查。通过与 GH27 成员的结构同源性推断 TmGalA 中的催化残基，以促进定点突变体的设计。产品分析证实野生型 (WT) 保留异头立体化学，类似于 GH27 酶。通过 D327G 和 D387G 突变酶的动力学分析、叠氮化物拯救以及叠氮化物拯救产物的测定，确认了保守的酸性残基。 Asp327 突变为 Gly 产生的突变体对芳基半乳糖苷的催化速率相对于 WT 酶低 200-800 倍。使用 D327G 酶的叠氮化物救援实验显示，与不使用叠氮化物的情况相比，催化速率高出 30 倍。向反应中添加叠氮化物导致叠氮化物 β-d-吡喃半乳糖苷的形成，证实 Asp327 是亲核残基。 Asp387Gly 突变的催化速度比对硝基苯基 α-d-吡喃半乳糖苷上的 WT 酶慢 1500 倍。不同 pH 值下的分析产生了 WT 酶的钟形曲线，但 D387G 随着 pH 值的增加表现出更高的活性。在叠氮化物存在下与 D387G 突变体的催化反应导致形成叠氮化物 α-d-半乳糖苷作为保留机制的产物。这些结果证实 Asp387 是 TmGalA 的酸/碱残基。此外，他们还表明 GH36 TmGalA 的生化特征与 GH27 酶密切相关，证实了 GH-D 家族成员的机制共性。
Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.