权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

定点突变与进化分子工程相结合对氨基酸脱氢酶进行功能修饰

基本信息

批准号：
15580079
负责人：
SAWA Yoshihiro
金额：
$ 2.3万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

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专一性的结构基础，并将这些知识应用于设计新的底物或辅因子专一性。用X射线结晶学方法测定了Phormidium loplideum天冬氨酸转氨酶的三维结构，为天冬氨酸结合部位的设计提供了依据。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。本研究克隆了枯草芽孢杆菌谷氨酸脱氢酶基因ROCG，并在大肠杆菌中进行了大量表达。纯化的重组BS-GluDH为六聚体结构(Mr 270 KDa)，对2-羟基戊二酸和L-谷氨酸具有严格的特异性，分别需要NADH和NAD^+作为辅因子。该酶热稳定性较差，Tm=41℃，这是由于六聚体的结构完整性较低所致。为了提高该酶的热稳定性，我们对克隆的谷氨酸脱氢酶进行了容易出错的聚合酶链式反应，引入了随机突变。从突变体库中分离到两个单突变酶Q144R和E27F，其Tm值分别为61℃和49℃。此外，Q144R在37℃的胺化反应中具有极高的k_&lt；CAT&gt；值(435 S)，是野生型的1.3倍。在预测与底物识别有关的残基中，枯草芽孢杆菌谷氨酸脱氢酶的Lys80、Gly82和Met101残基被突变为一系列单一突变体。G82K和M101S对草酸乙酯具有专一性，k值分别为3.45和5.68，分别是2-羟基戊二酸的265倍和473倍。我们正在利用分子进化工程来提高这些突变酶的K_&CAT&GT；值。