Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119234, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia.
Biochemistry (Mosc). 2020 Aug;85(8):920-929. doi: 10.1134/S0006297920080076.
Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.
与 OGDH 编码的 2-氧戊二酸脱氢酶（OGDH）不同，后者是所有动物组织中都存在的必需酶，DHTKD1 编码的同工酶 2-氧代己二酸脱氢酶（OADH）的表达取决于许多因素，并且突变 DHTKD1 表型很少表现出来。同工酶 2-氧戊二酸和 2-氧代己二酸都可以转化，这一事实也掩盖了 OADH 的生理意义。通过与其他 2-氧酸脱氢酶家族成员进行类比，可以假设 OADH 是催化 2-氧代己二酸氧化脱羧的多酶复合物的组成部分。本研究旨在对动物组织中的 OADH 进行分子特征分析。2-氧酸脱氢酶的系统发育分析表明，OADH 仅存在于动物和粘菌 Dictyostelium discoideum 中，与细菌 OGDH 位于同一分支中。通过免疫印迹和质谱法对部分纯化的动物 OADH 进行检查，鉴定出两种分子量约为 130 和 70 kDa 的 OADH 同工型。在细菌或酵母系统中表达人 DHTKD1 蛋白时，不会观察到这些同工型，在这些系统中，合成的 OADH 的分子量为预期值（约 100 kDa）。因此，动物组织中存在的 OADH 同工型可能是由于 DHTKD1 表达的动物特异性调节和/或编码蛋白的翻译后修饰所致。将在 OADH 制剂中鉴定出的肽映射到蛋白质结构上表明，70 kDa 同工型在 N 端截断，但保留了活性位点。由于 OGDH 的 N 端结构域是多酶复合物形成所必需的，因此 70 kDa 同工型可能催化不需要多酶结构的二羧酸 2-氧代酸的非氧化转化。在这种情况下，动物组织中 OADH 同工型的比例可能与 2-氧代己二酸的氧化和非氧化脱羧之间的比例相对应。
