A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7 T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

Abstract

ABSTRACT 3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (Acd DPN7 ) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7 T . During investigation of a Tn 5 :: mob -induced mutant defective in growth on 3,3′-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δ acd mutant was generated and verified the observed phenotype of the Tn 5 :: mob -induced mutant. For enzymatic studies, Acd DPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a:: acd DPN7 . The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO 3 2− ). Kinetic studies of Acd DPN7 revealed a V max of 4.19 μmol min −1 mg −1 , an apparent K m of 0.013 mM, and a k cat / K m of 240.8 s −1 mM −1 for 3SP-CoA. However, Acd DPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of Acd DPN7 and indicated a novel reaction mechanism. Hence, Acd DPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for the final desulfination step during catabolism of 3,3′-dithiodipropionate (DTDP), a sulfur-containing precursor substrate for biosynthesis of polythioesters.