Unveiling steps of the TDP degradation pathway in Variovorax paradoxus TBEA6

• Published in: Enzyme and microbial technology Vol. 160; p. 110095
• Main Authors: Schäfer, Lukas, Mikowsky, Nadine, Meinert-Berning, Christina, Steinbüchel, Alexander
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.10.2022
• Subjects: 3-Hydroxyisobutyryl-CoA hydrolase; 3-Hydroxypropionate dehydrogenase; 3′-Thiodipropionic acid; Degradation; Polythioester; PTE; TDP; Variovorax paradoxus TBEA6; PTE; 3-Hydroxyisobutyryl-CoA hydrolase; 3MB; TRAP; TDP 3; Tc; 3SP; Degradation; DTNB 5; TTT; 3MP; Tm; 3′-Thiodipropionic acid; Variovorax paradoxus TBEA6; HIB; TDP; Amp; DTDP 3; Polythioester; KU; 3 MV; PA; 3; CoA; Kan; 3HP; 3-Hydroxypropionate dehydrogenase; PET
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2022.110095

Since the role of biobased plastics increases every year, the search for alternatives to petrol-based polymers is very important. Variovorax paradoxus TBEA6 is able to grow with 3,3′-thiodipropionic acid (TDP) as sole source for carbon and energy. TDP can be used as a precursor substrate for the synthesis of polythioesters (PTE). To increase the feasibility of PTE synthesis, a good understanding of the degradation pathway of TDP in V. paradoxus TBEA6 is essential. Therefore, two putative 3-hydroxyisobutyryl-CoA hydrolases (VPARA_03110 & VPARA_05510) and two putative 3-hydroxypropionate dehydrogenases (VPARA_41140 & VPARA_54550) were investigated in this study. The deletion mutant V. paradoxus ∆VPARA_05510 showed a TDP-negative phenotype during growth experiments. The ability to grow with TDP as sole carbon source was successfully restored by complementation. Supernatant analysis revealed that the deletion mutant did not metabolize TDP or 3MP anymore. A specific enzyme activity up to 0.032 U/mg for the purified 3-hydroxyisobutyryl-CoA hydrolase VPARA_05510 was determined. A shift in the proteins (VPARA_54550) melting temperature of 6 °C with 2000 µM 3HP in comparison to protein without ligand was observed during thermal shift assays with the putative 3-hydroxypropionate dehydrogenase. •Variovorax paradoxus ∆VPARA_05510 did not growth with 3,3′-thiodipropionic acid.•Supernatant analysis revealed that the mutant did now consume TDP and 3MP any more.•Phenotype of the mutant was complemented using pBBRMCS-3::VPARA_05510 vector.•The reaction of 3-hydroxypropionyl-CoA to 3-hydroxypropionate could be verified.•Interaction of 3-hydroxypropionate dehydrogenases and the tested ligand were shown.