Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

Abstract

ABSTRACT The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act TBEA6 (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 Tn 5 :: 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 Tn 5 :: mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act TBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. Act TBEA6 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 V max 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 Act TBEA6 . For 3SP, the apparent V max was determined as 46.8 μmol min −1 mg −1 , which corresponds to a turnover number of 37.7 s −1 per subunit of Act TBEA6 . The apparent K m values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δ act mutant did not reproduce the phenotype of the Tn 5 :: 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 Tn 5 :: mob -induced mutant with pBBR1MCS5:: acd DPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn 5 :: mob transposon on acd TBEA6 , located downstream of act TBEA6 .