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谷氨酸脱氢酶突变体G82K和M101S对草酰乙酸酯的专一性，Kcat值分别为3.45和5.68 S，分别是2-羟基戊二酸的265倍和473倍。以新近获得的NAB依赖的耐热突变株GluDHQ144R为模板，构建了一系列双突变体酶，将底物专一性从2-氧戊二酸改变为草酰乙酸酯。在还原胺化反应中，GluDHs双突变株Q144R/G82K和Q144R/M101S表现出较高的选择性，有利于草酰乙酸酯产生天冬氨酸。Q144R/G82K和Q144R/M101S的Kcat值分别为199和2.14 S^-1和2.14 S^-1；丙酮酸分别为5.73和2.69。ECMDH是唯一一种…更多表现出轻微的L-天冬氨酸脱氨酶活性。分析比较了ECMDH和AAPDH的生化指标。在37-50℃范围内，L-天冬氨酸氧化脱氨的ECMDH的INC值高于AfAspDH值，Km值也远高于AfAspDH值。草酰乙酸酯(OAA)对AfAspdH的Km值随共存铵浓度的不同而变化。Lb提高了EcMDH的AspdH活性，尝试通过MOE-MD计算设计新的突变体。构建了多种EcMDH环突变体(N119S、N119A、A80P、P83V、G84V、D86G、R87G、S222G、V213F、V214F、E215D、E215R)，它们都具有很强的AspDH活性。突变体E215D和E215R对L-天冬氨酸脱氨酶的k值高于野生型，但Km值也远高于野生型。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必要的。较少

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