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（Bs-GluDH）的突变体G82 K和M101 S对草酰乙酸具有专一性，其Kcat值分别为3.45和5.68 s ~（-1）<-1>，比2-酮戊二酸的Kcat值分别高265和473倍。利用最近完成的NAB依赖性热稳定单突变体GluDH Q144 R作为模板，构建了一系列双突变体酶，以努力将底物特异性从2-酮戊二酸改变为草酰乙酸。在还原胺化反应中，在双突变体GluDH中，Q144 R/G82 K和Q144 R/M101 S显示出更高的草酰乙酸产生天冬氨酸的偏好。Q144 R/G82 K和Q144 R/M101 S对草酰乙酸和丙酮酸的Kcat值分别为199和2.14 s <-1>~（-1），5.73和2.69 s <-1>~（-1）。EcMDH是唯一一种 ...更多信息 对EcMDH和古生菌AspDH（AfAspDH）的生化指标进行了比较。在37-50℃范围内，EcMDH对L-天冬氨酸氧化脱氨的Inc值高于AfAspDH，Km值也远高于AfAspDH。AfAspDH对草酰乙酸（OAA）的Km值随共存铵离子浓度的变化而变化。lb提高了EcMDH的AspDH活性，尝试通过莫伊MD计算设计新的突变体。构建了各种EcMDH环突变体（N119 S、N119 A、A80 P、P83 V、G84 V、D86 G、R87 G、S222 G、V213 F、V214 F、E215 D、E215 R）以具有强AspDH活性。突变体E215 D和E215 R显示出比野生型更高的L-Asp脱氨基的k值，尽管Km值也比野生型高得多。然而，这两个突变体没有表现出还原胺化活性的OAA。因此，有必要应用分子进化工程技术来提高EcMDH的天冬氨酸脱氢酶活性。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