Serov Alexander E, Popova Anna S, Fedorchuk Vladimir V, Tishkov Vladimir I
Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 Moscow, Russian Federation.
Biochem J. 2002 Nov 1;367(Pt 3):841-7. doi: 10.1042/BJ20020379.
A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD(+) to NADP(+) has been constructed by introducing two single-point mutations, Asp(196)-->Ala (D196A) and Tyr(197)-->Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD(+)-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp(196) and Tyr(197) mediate the absolute coenzyme specificity of SceFDH for NAD(+).
通过引入两个单点突变,即天冬氨酸(196)突变为丙氨酸(D196A)和酪氨酸(197)突变为精氨酸(Y197R),构建了一种底物特异性从NAD⁺转变为NADP⁺的真核甲酸脱氢酶(EC 1.2.1.2,FDH)。诱变是基于以假单胞菌属101 FDH(PseFDH)晶体结构为模板对酿酒酵母NAD⁺特异性FDH(SceFDH)进行同源建模的结果。所得的模型结构表明,天冬氨酸(196)和酪氨酸(197)介导了SceFDH对NAD⁺的绝对辅酶特异性。