Functional modification of dehydrogenaws by the combination of evolutionary molecular engineering and molecular dynamics cal calculation

进化分子工程与分子动力学计算相结合的脱氢功能修饰

基本信息

批准号：
18580093
负责人：
SAWA Yoshihiro
金额：
$ 2.43万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Recently we have reported that the mutants, G82K and M101S of roc G glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) , were specific for oxaloacetate with Kcat values 3.45 and 5.68 s^<-1>, which were 265 and 473 folds higher than those for 2-oxoglutarate, respectively. Using recently accomplished NAB-dependent thermostable single mutant GluDH Q144R, as a template, a series of double mutant enzymes was constructed in the effort to change the substrate specificity from 2-oxoglutarate to oxaloacetate. In the reductive amination reaction, among the double mutant GluDHs Q144R/G82K and Q144R/M101S showed higher; preferences for oxaloacetate producing aspartate. The Kcat values of Q144R/G82K and Q144R/M101S were 199 and 2.14 s^<-1> respectively for oxaloacetate, and 5.73 and 2.69 s^<-1> respectively for pyruvate.The malate dehydrogenases (MDH) have been cloned and characterized from Escherichia coli, Bacillus subtilis and Nostoc sp.PCC7120. The EcMDH was the only dehydrogenase which … More showed slightly L-aspartate deamination activity The comparison of biochemical parameters between EcMDH and archeal AspDH (AfAspDH) were analyzed. The Inc value of EcMDH for L-Asp oxidative deamination was higher than that of AfAspDH at 37-50℃, while Km value was also much higher than that of AfAspDH. The Km value of AfAspDH for oxaloacetate (OAA) changed depending on coexistent ammonium concentrations. lb improve AspDH activity of EcMDH, new mutants were tried to be designed by using MOE MD calculation. Various EcMDH loop mutants (N119S, N119A, A80P, P83V, G84V, D86G, R87G, S222G, V213F, V214F, E215D, E215R) were constructed to have a strong AspDH activity. Mutants E215D and E215R showed higher k, values for L-Asp deamination than wild type although Km value was also much high than wild type. However, these two mutants showed no reductive amination activity for OAA. It is necessary to apply the molecular evolutionary engineering techniques for improving the aspartate dehydrogenase activity of EcMDH. Less

最近，我们有来自枯草芽孢杆菌（BS-Gludh）的突变体，g82k和M101s的ROC谷氨酸脱氢酶，其KCAT值3.45和5.68 s^<-1>的突变体，比2-265和473倍的KCAT值3.45和5.68 s^<-1>。使用最近完成的NAB依赖性热稳定的单个突变体Gludh Q144R的氧气变化是在双重突变剂量Q1144R/M101S中，使用了NAB依赖的热稳定性单个突变体Q144R Q144R Q144R的变化Q144R/G82K和Q144R/M101S分别为OxaloAcetate，2.14 s^<-1>分别对丙酮酸的5.73和2.69 s^<-1>分别为丙酮酸。 .pcc7120。 afaspDH的值也更大。 G84V，D86G，R87G，S222G，V214F，E215D，E215R）MUTS ASPDH活性。用于改善ECMDH天冬氨酸脱氢酶活性的麻烦技术