Functional modification of amino-acid dehydrogenases by the combination of site-directed mutagenesis and evolutionary molecular engineering

我们研究的主要目标之一是了解氨基酸脱氢酶中氨基酸底物特异性的结构基础，并将这些知识应用于新型底物或辅因子特异性的工程中。用x射线晶体学方法测定了天冬氨酸转氨酶的三维结构，为天冬氨酸结合位点的设计提供了依据。选择丙氨酸脱氢酶基因和谷氨酸脱氢酶基因作为诱变模板。从枯草芽孢杆菌中克隆了编码谷氨酸脱氢酶的rocG基因（Bs-GluDH），并在大肠杆菌中大量表达。重组Bs-GluDH经纯化达到均匀性，确定为六聚体结构（M_r 270 kDa），对2-氧葡萄糖酸酯和l -谷氨酸具有严格的特异性，分别需要NADH和NAD^+作为辅助因子。在T_m=41℃时，由于六聚体的结构完整性较低，酶表现出较低的热稳定性。为了提高该酶的热稳定性，我们对克隆的GluDH进行了容易出错的PCR，引入随机诱变。从突变体文库中分离到两个单突变酶Q144R和E27F，其T_m值分别为61℃和49℃。Q144R在37℃的胺化反应中具有较高的k_<cat>值（435 s^<-1>），是野生型的1.3倍。在预测的涉及底物识别的重要残基中，枯草芽孢杆菌谷氨酸脱氢酶的Lys80、Gly82和Met101残基突变为一系列单突变体。野生型酶对2-氧葡萄糖酸酯的k_<cat>值为344 s^<-1>，而G82K和M101S对草酰乙酸酯的k_<cat>值为3.45和5.68 s^<-1>，分别是2-氧葡萄糖酸酯的265倍和473倍。我们正在利用分子进化工程技术提高这些突变酶的k_<cat>值。

One of the major goals of our studies has been to understand the structural basis of amino acid substrate specificity in amino acid dehydrogenase, and to apply such knowledge to the engineering of novel substrate or cofactor specificities. The three dimensional structure of aspartate aminotransferase from Phormidium lapideum has been determined for design of aspartate binding site by X-ray crystallography. The alanine dehydrogenase gene and glutamate dehydrogenase gene were chosen as templates for the mutagenesis. The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in E.coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined as a hexameric structure (M_r 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate requiring NADH and NAD^+ as cofactors, respectively. The enzyme showed low thermostability with T_m=41℃ due to low structural integrity of hexamer. To improve thermal stability of this enzyme, we performed error-prone PCR introducing random mutagenesis on cloned GluDH. Two single mutant enzymes Q144R and E27F were isolated from mutant library whose T_m values were 61℃ and 49℃, respectively. Furthermore, Q144R has a remarkably high k_<cat> value (435 s^<-1>) for amination reaction at 37℃ which is 1.3 times higher than that of wild type. Among the residues predicted to be important involving substrate recognition, Lys80,Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild type enzyme is specific for 2-oxoglutarate with k_<cat> value 344 s^<-1>, whereas G82K and M101S are specific for oxaloacetate with k_<cat> values 3.45 and 5.68 s^<-1>, which are 265 and 473 folds higher than those for 2-oxoglutarate, respectively. We are in progress to enhance the k_<cat> values of those mutant enzymes by using molecular evolutional engineering.