Enzymes of the microbial catabolism of the thioether 3,3´-thiodipropionic acid
Microbial Ecology and Applied Microbiology
Final Report Abstract
The project aimed at the investigation of the microbial degradation of thioester 3,3´‐thiodipropionic acid (TDP) by the Gram‐negative bacterium Variovorax paradoxus TBEA6. Although most of the enzymes participating in the catabolism of the organic sulfur compound were identified previously to this project, only one out of four initially proposed reactions was confirmed biochemically. The main goal of this project was the investigation of the other steps of the TDP catabolism. The initially proposed reactions were successfully verified or refuted throughout the funding period. The first degradation step in the catabolic pathway of TDP was suggested to be catalyzed by an FADdependent oxidoreductase (Fox). It was a major goal of this project to investigate the catalytic activity of Fox. Thereby, it was shown that the enzyme is a 2‐D‐hyroxyacid dehydrogenase (Meinert et al.2018). Hence, the enzyme is not catalyzing the enzymatic cleavage of TDP to 3‐mercaptopropionic acid (3MP) and 3‐hydroxypropionic acid as it was assumed. Prior to this study, 3MP was shown to be an intermediate of the catabolic pathway of TDP. Its degradation is achieved by the oxidation of 3MP to 3SP, catalyzed by the 3MP dioxygenase. This is the only reaction, which was verified prior to this project. Next, 3SP is activated either by an acyl‐CoA transferase (ActTBEA6) or a succinyl‐CoA Transferase (SucCDTBEA6) of which the strain has two copies. By the extensive study of homologs of SucCDTBEA6, it was shown that the catalyzed activation of 3SP to 3SP‐CoA is not a unique function of only a few SucCDs. Therefore, the biochemical characterization of the copies from V. paradoxus TBEA6 was not conducted. Subsequently, 3SP‐CoA is desulfinated by the desulfinase AcdATBEA6. This reaction was verified during the progress of this project and kinetic data were determined during the biochemical characterization. The results on a study with different Acds showed that only the desulfinases of Advenella mimigardefordensis strain DPN7, V. paradoxus TBEA6, Bacillus xenovorans LB400 and Cupriavidus necator N‐1 were able to catalyze this reaction and that this novel enzyme evolved from acyl‐CoA dehydrogenases (Schürmann et al. 2014). Because FoxTBEA6 does not catalyze the initial cleavage of TDP, additional strategies were followed to identify proteins involved in the degradation of the organic sulfur compound. The availability of the complete genome of V. paradoxus TBEA6 enabled the application of 2‐dimensional SDS‐polyacrylamide gel electrophoresis. Thereby, two enoyl‐CoA hydratases and a putative dehydrogenase showed increased abundance during cultivation of V. paradoxus TBEA6 with TDP in comparison to the controls. Deletion of these genes evoked restricted growth with TDP or inhibited growth of the mutants completely. It is noticeable that 3SP, which is one of the intermediates of TDP degradation, could still be consumed by the mutants. Enoyl‐CoA hydratases belong to the family of the crotonases. The substrates of these enzymes are unexceptional CoA‐esters. Therefore, TDP‐CoA was synthesized by our laboratory, and it was shown that the compound is the ligand of one of the enoyl‐CoA hydratase. These findings were unexpected and indicated that enzymes normally found in fatty acid metabolism are involved in the initial degradation steps of TDP. The investigation of the detailed reaction mechanisms will be very interesting and part of a future project.
Publications
- Identification of 3-sulfinopropionyl coenzyme a (CoA) desulfinases within the acyl-CoA dehydrogenase superfamily. Journal of Bacteriology, Vol. 196. 2014, issue 4, pp. 882–893.
Schürmann, M., Demming, R. M., Krewing, M., Rose, J., Wübbeler, J. H., Steinbüchel, A.
(See online at https://doi.org/10.1128/JB.01265-13) - 3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis DPN7T: Crystal structure and function of a desulfinase with an acyl-CoA dehydrogenase fold. Acta Crystallographica Section D: Biological crystallography, Vol. 71. 2015, pp. 1360–1372.
Schürmann, M., Meijers, R., Schneider, T. R., Steinbüchel, A. & Cianci, M.
(See online at https://doi.org/10.1107/S1399004715006616) - The genome of Variovorax paradoxus strain TBEA6 provides new
understandings for the catabolism of 3,3’-thiodipropionic acid and hence the production of polythioesters. Journal of Biotechnology, Vol. 209. 2015, pp. 85-95.
Wübbeler, J. H., Hiessl, S., Meinert, C., Poehlein, A., Schuldes, J., Daniel, R., Steinbüchel, A.
(See online at https://doi.org/10.1016/j.jbiotec.2015.06.390) - The unexpected function of a putative Flavin-dependent oxidoreductase (Fox) from Variovorax paradoxus TBEA6. FEMS Microbiology Letters, Vol. 365. 2018, Issue 6, fny011.
Meinert, C., Schürmann, M., Domeyer, J.-E., Poehlein, A., Daniel, R., Steinbüchel, A.
(See online at https://doi.org/10.1093/femsle/fny011) - The catabolism of 3,3’-thiodipropionic acid in Variovorax paradoxus strain TBEA6: A proteomic analysis. PLoS One, Vol. 14. 2019, Issue 2: e0211876.
Viktoria Heine, Christina Meinert-Berning, Janina Lück, Nadine Mikowsky, Birgit Voigt, Katharina Riedel, Alexander Steinbüchel
(See online at https://doi.org/10.1371/journal.pone.0211876)
