II类NAD-依赖型异柠檬酸脱氢酶的功能与进化机制研究

NAD-dependence is an ancestral phenotype of isocitrate dehydrogenase. All known type II IDHs are NADP-dependent (NADP-IDH), and no type II NAD-dependent IDHs (NAD-IDH) have been reported yet. In this study, we are aiming at finding and characterizing type II NAD-IDH. Firstly, bioinformatic analysis will be applied to search for type II NAD-IDH, and the phylogenetic tree of IDH protein family will be re-constructed using molecular phylogeny to confirm the evolutionary position of type II NAD-IDH. Next, typical novel type II NAD-IDHs will be heterologously expressed and their enzymatic properties will be determined. Then, the alteration of the coenzyme specificity of type II IDH from NAD (ancestral phenotype) to NADP (adaptive phenotype) will be carried out using structural-based multiple sequence alignment and protein engineering. Fourthly, the recombinant strains containing the wild-type and mutated type II NAD-IDH will be constructed using chromosomal homologous recombination. The adaptive advantages of all recombinant strains can be compared and analyzed through the growth rate of them on the different carbon sources. This project will prove the mechanism of functional evolution that the NADP-dependence of type II IDH is evolved from the ancestral NAD-dependent phenotype.

NAD-依赖型是异柠檬酸脱氢酶(IDH)的古老表型，已知的II类IDH都是NADP-依赖型(NADP-IDH)，还未发现具有古老表型的II类NAD-依赖型IDH (NAD-IDH)。本项目拟通过生物信息学分析找到II类NAD-IDH，再应用分子系统学方法重建IDH的系统进化树，确定II类NAD-IDH的进化位置。接着对新发现的代表性II类NAD-IDH进行原核表达以及酶学性质鉴定。然后应用基于结构的氨基酸序列比对和蛋白质工程技术，实现II类NAD-IDH的辅酶特异性由NAD(古老表型)向NADP(适应表型)的转换。再应用同源重组技术分别构建包含野生型和突变体II类NAD-IDH编码基因的重组大肠杆菌，通过测定在不同碳源中的生长速率，比较重组菌株的适应优势，证明II类IDH的辅酶特异性从古老的NAD-依赖型演化为NADP-依赖型的功能进化机制。

异柠檬酸脱氢酶(isocitrate dehydrogenase, IDH)可以分为I型IDH和II型IDH两大亚家族。I型IDH既有NADP+依赖型(NADP-IDH)，也有NAD+依赖型(NAD-IDH)， 而目前所报道的II型IDH都是NADP+依赖型。本研究通过检索GenBank数据库中大量的IDH序列信息，成功找到了两类新型的II型NAD-IDH。第一类新型NAD-IDH主要来源于海洋真核藻类，以Ostreococcus lucimarinus IDH (OlIDH)和Micromonas sp. RCC299 IDH (MiIDH)为代表。系统进化分析表明，本研究新发现的这类新型NAD-IDH非常清晰的与II型IDH聚在同一进化枝，但独立聚集在一个进化枝。而与II型NADP-IDH相比，新型IDH的辅酶结合位点氨基酸完全不同，提示其可能为NAD依赖型。.酶学研究表明，OlIDH和MiIDH在溶液中同为同源二聚体。酶动力学参数测定分析表明，OlIDH和MiIDH对于NAD的偏好性分别是NADP的99倍和224倍，从而证明本研究所发现的新型IDH确实都为NAD依赖型。酶活测定和性质研究发现，OlIDH的催化活性依赖于二价金属离子的存在，Mn2+是其最适激活剂。OlIDH的最适pH是8.5，最适反应温度是40 oC，在45 oC时，酶的热稳定性最强。第二类新型IDH为单体型NAD-IDH，以来源于海洋细菌Campylobacter sp. FOBRC14的IDH (CaIDH)代表。CaIDH在溶液中为单体状态。酶动力学参数测定分析表明，CaIDH对于NAD的偏好性分别是NADP的61倍。酶活测定和性质研究发现，CaIDH的催化活性依赖于二价金属离子的存在，Mn2+是其最适激活剂。Mn2+存在时，CaIDH的最适pH是8.0，最适反应温度是45 oC，在45 oC时，酶的热稳定性最强。.采用定点突变技术，将CaIDH的辅酶结合位点（Leu584，Asp595）突变为单体型NADP-IDH相应的辅酶结合位点氨基酸残基（His和Arg）。动力学分析显示，通过关键位点的突变，将CaIDH的辅酶依赖性由NAD改造成了NADP。我们的结果初步证明，当细菌在贫瘠碳源上生长时，单体NAD-IDH可以替代二聚体NADP-IDH发挥功能。