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

该项目研究了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。先前分离的变异变形杆菌菌株TBEA6是阐明TDP分解代谢的最有利候选菌株；它作为一种模式生物，在这个项目中被使用。大多数基因及其翻译产物，被认为参与了TDP的分解代谢途径，我们的实验室之前已经确定了这些基因及其翻译产物。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催化该途径的第二步，将3-巯基丙酸（3MP）的巯基氧化为亚砜基，从而形成3-亚砜丙酸盐。然而，重要的初始步骤，即TDP转运到细胞中并随后将TDP切割成3-羟基丙酸和3MP，目前还只是假设。假设的输运系统属于TTT （Tripartite Tricarboxylate transport）家族。据推测，负责TDP裂解的酶是一种新型的fad依赖性氧化还原酶。这些反应是这个项目的主要焦点。此外，本文还详细研究了在CoA连接酶的催化下，将中间体3-亚砜丙酸活化成3-亚砜丙酰辅酶a，以及随后由酰基辅酶a脱氢酶样脱硫酶进行的脱硫反应。因此，在这个项目中，体外和体内酶分析将被开发和优化(1)。此外，V. paradoxus菌株TBEA6的全基因组DNA由Göttingen Genomics Laboratory （G2L）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学研究（iii）。研究结果将用于增强基于无毒前体TDP的聚硫酯的生物技术生产。

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.