The unexpected function of a Flavin-dependent oxidoreductase from Variovorax paradoxus TBEA6

3,3'-Thiodipropionic acid (TDP) is used as an additive in food and cosmetic industry and as precursor substrate for biotechnical polythioester production. Its catabolism was investigated in Variovorax paradoxus TBEA6 previous to this study. It was reported that the insertion of the transposon Tn5::mob into a gene showing high homology to flavin-dependent oxidoreductases (fox) resulted in impaired growth with TDP. Therefore, it was assumed that the initial cleavage of TDP is catalyzed by an FAD-dependent oxidoreductase (Fox, VPARA_05580). Accordingly, fox was heterologously expressed as a thioredoxin fusion protein. Analytical size exclusion chromatography revealed a homodimeric structure and the presence of the cofactor FAD. In vitro experiments showed that FoxTBEA6 is a D-2-hydroxy acid specific dehydrogenase and that its activity is enhanced in presence of either Ni2+, Co2+ or Zn2+. Cleavage of TDP by FoxTBEA6 was not observed. The findings are contrary to restricted growth with TDP of the transposon mutants and the previously published deletion mutant V. paradoxus TBEA6 Δfox. In this study, this contradiction was investigated by generation of additional deletion mutants, in which partial or complete deletion of fox did not affect utilization of TDP, and the mapping of single nucleotide polymorphisms (SNPs) in V. paradoxus TBEA6 Δfox.