The catabolism of 3,3’-thiodipropionic acid in Variovorax paradoxus strain TBEA6: A proteomic analysis

Abstract

Variovorax paradoxus strain TBEA6 is one of the few organisms known to utilize 3,3’-thiodipropionate (TDP) as the only source of carbon and energy. It cleaves TDP to 3-mercaptopropionate (3MP), which is a direct precursor for polythioester synthesis. To establish this process in V. paradoxus TBEA6, it is crucial to unravel its TDP metabolism. Therefore, a proteomic approach with subsequent deletion of interesting genes in the bacterium was chosen. Cells were cultivated with D-gluconate, TDP or 3-sulfinopropionate as the only carbon sources. Proteins with high abundances in gels of cells cultivated with either of the organic sulfur compounds were analyzed further. Thereby, we did not only confirm parts of the already postulated TDP metabolism, but also eight new protein candidates for TDP degradation were detected. Deletions of the corresponding genes (two enoyl-CoA hydratases (Ech-20 and Ech-30), an FK506-binding protein, a putative acetolactate synthase, a carnitinyl-CoA dehydratase, and a putative crotonase family protein) were obtained. Only the deletions of both Ech-20 and Ech-30 led to a TDP negative phenotype. The deletion mutant of VPARA_05510, which encodes the putative crotonase family protein showed reduced growth with TDP. The three genes are located in one cluster with genes proven to be involved in TDP metabolism. Thermal shift assays showed an increased stability of Ech-20 with TDP-CoA but not with TDP. These results indicate that Ech-20 uses TDP-CoA as a substrate instead of TDP. Hence, we postulate a new putative pathway for TDP metabolism. Ech-30 interacts with neither TDP-CoA nor TDP but might interact with other CoA-activated intermediates of the proposed pathway. Further enzyme characterization is necessary to unravel the complete pathway from TDP to 3MP.

3,3′-Thiodipropionic acid (TDP), a possible precursor for the synthesis of polythioesters: identification of TDP transport proteins in Variovorax paradoxus TBEA6

“…Utilization of TDP by V. paradoxus TBEA6 was previously investigated and proved in our laboratory (Heine et al 2019;Brandt et al 2014;Schürmann et al 2013;Bruland et al 2009). TDP is first cleaved into 3-hydroxypropionate (3-HP) and 3-mercaptopropionate (3-MP), by an flavin adenine dinucleotide (FAD) linked oxidase (Fox) (Brandt et al 2014;Bruland et al 2009).…”

“…Succinyl-CoA and acetyl-CoA from all three pathways enter into central metabolism. Recently, proteomic analysis conducted by Heine et al, (2019) provided new insights in TDP metabolism. Even though tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli, it could not become a TDP utilizer, because E. coli does not produce the enzymes which can degrade TDP.…”

“…Subsequent removal of the sulfur moiety is catalyzed by a desulfinase, Acd, yielding sulfite, and propionyl-CoA. Propionyl-CoA enters in to central metabolism via three different possible pathways, i.e., the malonate semialdehyde pathway, the methylmalonyl-CoA pathway, and the methylcitrate cycle (Heine et al 2019 ). In the first pathway, dehydrogenases and hydratases convert the propionyl-CoA in to acetyl-CoA.…”

“…Schäfer et al ( 2019 ) conducted thermoshift assays using protein TctC1 with locus tag MIM-c39190 in A. mimigardefordensis DPN7T and reported that TctC1 showed a substantial shift in the melting temperature with citrate, whereas no shift occurred with the ligand α-ketoglutarate. Heine et al ( 2019 ) used TDP-CoA as ligand and the two proteins Ech-20 and Ech-30 from V. paradoxus TBEA6 in thermal shift assays to explain a protein ligand interaction. They reported that in presence of TDP-CoA a shift in the melting temperature of Ech-20 was obtained, but no shift was obtained with TDP.…”

“…Reports are available about growth of V. paradoxus strain TBEA6 using various substrates. Heine et al ( 2019 ) used the TDP, succinate, and gluconate each at 60 mM concentration as carbon source for growth of strain TBEA6. Strain TBEA6 grew also well with 30 mM TDP (Wübbeler et al 2015 ) and 20 mM sodium gluconate along with 1 mM TDP as carbon sources (Brandt et al 2014 ).…”