A novel 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis strain DPN7T acting as a key enzyme during catabolism of 3,3'-dithiodipropionic acid is a member of the acyl-CoA dehydrogenase superfamily.
3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7(T). During investigation of a Tn5::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 Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. 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 (SO3(2-)). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 μmol min(-1) mg(-1), an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s(-1) mM(-1) for 3SP-CoA. However, AcdDPN7 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 AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 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.
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Marc
M
Schürmann
Anika
A
Deters
Jan Hendrik
JH
Wübbeler
Alexander
A
Steinbüchel
D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042401:Alcaligenaceae / Q000201:enzymology* / Q000378:metabolism*; D019117:Burkholderia / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D011422:Propionates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D003067:Coenzymes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011422:Propionates; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C013298:thiodipropionic acid; D042964:Acyl-CoA Dehydrogenase