Succinyl-CoA:3-sulfinopropionate CoA-transferase from Variovorax paradoxus strain TBEA6, a novel member of the class III coenzyme a (CoA)-transferase family

Abstract

The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, ActTBEA6 (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoAtransferases. In the present study, analyses of the translation product clearly allocated ActTBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. ActTBEA6 was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96+3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent Vmax of 44.6 μmol min-1 mg-1 for succinyl-CoA, which corresponds to a turnover number of 36.0 s-1 per subunit of ActTBEA6. For 3SP, the apparent Vmax was determined as 46.8 μmol min-1 mg-1, which corresponds to a turnover number of 37.7 s-1 per subunit of ActTBEA6. The apparent Km values were 0.08mMfor succinyl-CoA and 5.9mMfor 3SP. Nonetheless, the V. paradoxus δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mobinduced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acdTBEA6, located downstream of actTBEA6. © 2013, American Society for Microbiology.