海洋微型生物异柠檬酸脱氢酶的功能鉴定及进化机制研究

The protein family of isocitrate dehydrogenase (IDH) is an essential enzyme family in the organisms. However, no NAD-dependent IDH (ancestral protein) has been found in a monophyletic group, type II subfamily. A large number of genomes from marine organisms have been sequenced, which become a breakthrough for completing the IDH family. Many methods will be employed to study the novel IDHs, including molecular phylogenetics, functional genomics, enzymology, structural biology and protein engineering. The phylogenetic tree of IDH family will be rebuilt, and the functions of IDHs from marine bacteria and algae in the three new clades will be identified. Based on the alteration of specificity, it will be detected that whether the marine bacterial NAD-IDH is the ancestor of the bacterial NADP-IDH and whether the marine algae NAD-IDH is the ancestor of the eukaryotic NADP-IDH in the same subfamily. It will be also detected that whether the marine algae monomeric NADP-IDH is evolved from the bacterial monomeric NAD-IDH in the same subfamily. Considering that the evolutionary positions of the marine algae monomeric NADP-IDH and the bacterial monomeric NADP-IDH are crossed, the possible horizontal gene transfer between them will be analyzed. Based on the research of IDH phosphorylation mechanism, the phylogenetic path of the glyoxylate cycle will be explored to provide the evidences for the horizontal gene transfer among the bacteria, the plants and the animals. The investigation of NAD-IDH from the marine microbes in the new clades will fill the gap of the phylogenetic study of IDH family.

异柠檬酸脱氢酶(IDH)家族是重要的酶家族，但单起源的IDH第II亚族至今未发现祖先蛋白NAD-IDH。大量涌现的海洋生物基因组序列成为补齐IDH家族的突破口。本项目拟采用分子系统学、功能基因组学、酶学、结构生物学、蛋白质工程等技术，重建IDH家族系统发生树；对3个新进化分支中的海洋藻类及细菌IDH进行功能鉴定；基于辅酶依赖性转换实验，检测新的海洋细菌NAD-IDH是否是本亚族细菌NADP-IDH的祖先、海洋藻类NAD-IDH是否是本亚族真核NADP-IDH的祖先、及海洋藻类单体NADP-IDH是否由单体NAD-IDH演化而来；鉴于海洋藻类单体NADP-IDH与细菌单体NADP-IDH交叉的进化位置，分析二者间可能的基因水平转移关系；结合IDH磷酸化机制的研究，探寻乙醛酸循环的进化路径，为可能的细菌、植物、动物之间的基因水平转移提供证据。本研究将填补IDH家族系统分类研究中缺少的重要一环。

异柠檬酸脱氢酶(IDH) 蛋白家族是所有生物体中心代谢中的关键酶，与能量代谢、氨基酸和维生素合成、氧化应激以及多种类型癌症的发生发展密切相关。本项目完成了对IDH家族4个新进化分支中的海洋藻类或细菌IDH的酶学鉴定，分别是来自Type II亚家族的海仙菌NAD-HsIDH、长盐单胞菌NAD-HeIDH及海滨聚集杆菌NAD-ClIDH，以及海洋青绿藻NAD-OtIDH、三角褐指藻NAD-PtIDH1及蓝隐藻NAD-GtIDH，和来自Type III亚家族的三角褐指藻NADP-PtIDH2和甲基拟甲烷球菌NADP-MmIDH。HsIDH、HeIDH及ClIDH是同源六聚体NAD-IDHs，OtIDH和PtIDH1是藻类同源二聚体NAD-IDHs，PtIDH2是单体型同源四聚体NADP-IDH，这些IDH均是此前未见报道的酶。辅酶依赖性转换实验，为Type II亚家族真核NADP-IDH、Type II亚家族细菌NADP-IDH、以及Type III亚家族藻类和古菌NADP-IDH找到了可能的祖先酶。结合系统发生树分析，探讨了海洋藻类与细菌之间可能的基因水平转移关系。解析了5种IDH晶体结构，包括OtIDH (PDB ID: 6IXL, 6IXT, 6IXN, 6IXU)、XcIDH (PDB ID: 6LTF)、McIDH (PDB ID: 6LKY)、PtIDH1 (PDB ID: 6LKZ)以及AbIDH2 (PDB ID: 5Z16)。本项目实施前，Type II 亚家族所有IDH均为NADP-IDH，PDB里除了真核线粒体异源寡聚体NAD-IDH的结构，只有一种细菌NAD-IDH结构。本项目实施后，4个新进化分支填补了Type II IDH和Type III亚家族系统分类中缺失环节。同时不同NAD-IDH结构的解析，也极大的丰富了PDB中NAD-IDH的结构信息。上述成果对于更好的了解IDH蛋白家族的生理功能具有重要的科学意义。

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