Enzymes of the microbial catabolism of the thioether 3,3´-thiodipropionic acid

拟议项目研究有机硫化合物和硫醚3，3 ′-硫代二丙酸（TDP）作为唯一碳源的罕见微生物降解。先前分离的β-变形杆菌Variovorax paradoxus菌株TBEA 6是阐明TDP的TDP催化作用的最有利候选者;它作为模式生物并用于本项目。本实验室已鉴定了TDP代谢途径中的大部分基因及其翻译产物。该途径的关键酶3-巯基丙酸双加氧酶（Mdo）的详细生化表征已经完成。MDO催化所提出的途径的第二步，即3-巯基丙酸酯（3 MP）的巯基氧化成亚磺基，从而形成3-亚磺基丙酸酯。然而，重要的初始步骤，即。e. TDP转运到细胞中以及随后将TDP裂解成3-羟基丙酸和3 MP还只是推测。假设的运输系统属于TTT（三组分三羧酸盐运输）家族。负责TDP裂解的酶pueridine是一种新的FAD依赖性氧化还原酶。这些反应是本项目的主要焦点。此外，将详细研究所提出的将中间体3-亚磺基丙酸活化为3-亚磺基丙酰辅酶A（由辅酶A连接酶催化）以及随后由酰基辅酶A酰辅酶A酶样辅酶A酰辅酶A酶进行的酰辅酶A化反应。因此，在本项目期间，将开发和优化体外和体内酶测定（i）。此外，哥廷根基因组学实验室（G2 L）对奇异弧菌菌株TBEA 6的全基因组DNA进行了测序，并将进行处理和注释（ii）。随后，将完成TDP和替代碳源生长过程中的蛋白质组研究（iii）。研究结果将用于基于无毒前体TDP的聚硫酯的增强生物技术生产。

The proposed project investigates the rare microbial degradation of the organic sulfur compound and thioether 3,3´-thiodipropionic acid (TDP) as sole carbon source. The previously isolated beta-proteobacterium Variovorax paradoxus strain TBEA6 is the most favourable candidate to elucidate TDP catabolism of TDP; it serves as a model organism and is employed in this project. Most genes and their translational products, which are putatively involved in the catabolic pathway of TDP, were previously identified by our laboratory. A detailed biochemical characterization of the key enzyme of this pathway, the 3-mercaptopropionate dioxygenase (Mdo), was already done. Mdo catalyzes the second step of the proposed pathway, the oxidation of the sulfhydryl group of 3-mercaptopropionate (3MP) into a sulfino group, thereby forming 3-sulfinopropionate. However, the important initial steps, i. e. the transport of TDP into the cell and its subsequent cleavage of TDP into 3-hydroxypropionic acid and 3MP, are only suppositions, yet. The assumed transport system belongs to the TTT (Tripartite Tricarboxylate Transport) family. The enzyme putatively responsible for the cleavage of TDP is a novel FAD-dependent oxidoreductase. These reactions are in the major focus of this project. In addition, the proposed activation of the intermediate 3-sulfinopropionic acid into 3-sulfinopropionyl-CoA, which is catalyzed by a CoA ligase, and the subsequent desulfination reaction performed by an acyl-CoA dehydrogenase-like desulfinase will be investigated in detail. Thus, during this project in vitro and in vivo enzyme assays will be developed and optimized (i). Moreover, the whole-genome DNA of V. paradoxus strain TBEA6 was sequenced by the Göttingen Genomics Laboratory (G2L), and will be processed and annotated (ii). Subsequently, proteome studies during growth on TDP and alternative carbon sources will be accomplished (iii). The findings will be used for the enhanced biotechnological production of polythioesters based on the nontoxic precursor TDP.