Bobrov K S, Borisova A S, Eneyskaya E V, Ivanen D R, Shabalin K A, Kulminskaya A A, Rychkov G N
Petersburg Nuclear Physics Institute, Orlova Roscha, Gatchina, 188300, Leningrad Region, Russia.
Biochemistry (Mosc). 2013 Oct;78(10):1112-23. doi: 10.1134/S0006297913100052.
At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.
在高浓度对硝基苯基-α-D-吡喃半乳糖苷（pNPGal）作为底物的情况下，嗜热栖热菌（Thermotoga maritima）的α-半乳糖苷酶（TmGalA）催化其水解时会伴随转糖基化反应，生成（α1,2）-、（α1,3）-和（α1,6）-对硝基苯基（pNP）-二半乳糖苷的混合物。对该酶活性位点内pNP-二半乳糖苷形成之前的反应阶段进行分子建模，揭示了预期修饰后能提高转糖基化效率的氨基酸残基。对预测的氨基酸残基替代进行定点诱变后，编码野生型TmGalA及其突变体的基因在大肠杆菌中表达，分离出相应的酶，并检测其在合成不同pNP-二半乳糖苷时的转糖基化活性。结果表明，与野生型酶相比，三个突变体F328A、P402D和G385L的总转糖基化作用显著增强。此外，F328A突变体能够产生具有（α1,2）键的区域异构体，其产量比野生型TmGalA高16倍。
