Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7 T during Degradation of 3,3′-Dithiodipropionic Acid

Abstract

ABSTRACT The sucCD gene of Advenella mimigardefordensis strain DPN7 T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn 5 :: mob- induced mutants of A. mimigardefordensis strain DPN7 T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis Δ sucCD . These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7 T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD Am ) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD Am is Mg 2+ or Mn 2+ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate ( V max = 9.85 ± 0.14 μmol min −1 mg −1 , K m = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% ( V max = 0.12 ± 0.01 μmol min −1 mg −1 ) and the affinity was 6-fold lower ( K m = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCD Am is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.