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

最近我们报道了枯草芽孢杆菌（Bacillus subtilis, Bs-GluDH）的roc G谷氨酸脱氢酶突变体G82K和M101S对草酰乙酸具有特异性，Kcat值分别为3.45和5.68 s^<-1>，分别是2-氧酰乙酸的265倍和473倍。以最近完成的nab依赖性耐热单突变体GluDH Q144R为模板，构建了一系列双突变体酶，试图将底物特异性从2-氧戊二酸转变为草酰乙酸。在还原胺化反应中，双突变GluDHs中Q144R/G82K和Q144R/M101S表现较高；草酰乙酸产生天冬氨酸的偏好。Q144R/G82K和Q144R/M101S对草酰乙酸的Kcat值分别为199和2.14 s^<-1>，对丙酮酸的Kcat值分别为5.73和2.69 s^<-1>。从大肠杆菌、枯草芽孢杆菌和Nostoc sp.PCC7120中克隆并鉴定了苹果酸脱氢酶（MDH）。EcMDH是唯一具有少量l -天冬氨酸脱氨活性的脱氢酶，并与原AspDH （AfAspDH）进行生化参数比较。37 ~ 50℃时，EcMDH对L-Asp氧化脱氨的Inc值高于AfAspDH， Km值也远高于AfAspDH。草酰乙酸（OAA）的AfAspDH Km值随共存铵浓度的变化而变化。为了提高EcMDH的AspDH活性，利用MOE - MD计算，尝试设计新的突变体。构建了多种具有较强AspDH活性的EcMDH环突变体（N119S、N119A、A80P、P83V、G84V、D86G、R87G、S222G、V213F、V214F、E215D、E215R）。突变体E215D和E215R的L-Asp脱胺k值高于野生型，Km值也高于野生型。然而，这两个突变体对OAA没有还原胺化活性。应用分子进化工程技术提高EcMDH的天冬氨酸脱氢酶活性是十分必要的。少

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