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

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 Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ⌬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 DPN7T 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 mol 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 mol 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.3,3Ј-Dithiodipropionic acid (DTDP) is an organic disulfide and a precursor for the production of polythioesters (PTEs) by bacteria (25). Further applications for DTDP are thermodynamic studies (40), development of secondary batteries (52), amino acid analysis (53), and the construction of self-assembling monolayers (10). Microbial production of PTEs from simple carbon sources and inorganic sulfur is currently not possible. Knowledge of the catabolism of organic sulfur compounds in bacteria could provide a reasonable strategy to engineer strains suitable for PTE production. A first step in this direction was the isolation of bacteria able to utilize DTDP as the sole source of carbon and energy. Advenella mimigardefordensis strain DPN7T , a betaproteobacterium, found in mature compost in a waste management facility was one of the isolates (15,56

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

“…This, with reference to published spectra (44,81,82), unambiguously showed that p3MDO readily oxidizes 3-MPA. Further evidence was provided by mass spectrometry (Ϫve ESI), which showed a m/z peak of 136.9918 that compared well with the calculated profile (136.9914) and the 3-SPA standard (136.9919).…”

“…Synthesis of the Disodium Salt of 3-SPA-3-SPA was synthesized based on the method by Jollès-Bergeret (68) and a modified version by Schurmann et al (44). All chemicals for the synthesis were used without further purification from SigmaAldrich.…”

“…Strains from the Pseudomonas family are capable of 3-MPA utilization (28). Conversion of 3-SPA to 3-sulfinopropionyl-CoA in Proteobacteria has been attributed to succinylCoA synthetase, (44,45); however, enzymatic oxidation of 3-MPA to 3-SPA remains to be identified. CDO homologues from bacteria (Ralstonia eutropha, Protein Data Bank accession number 4QMA (formerly 2GM6); Bacillus subtilis, 3EQE) are structurally very similar to mammalian CDO, for which several x-ray crystal structures are available, but the substitution of glutamine in bacterial enzymes for arginine at position 60 of the mammalian active site suggests altered substrate specificity.…”

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

“…In the next step, a thiol dioxygenase (Mdo) catalyzes the oxidation to 3-sulfinopropionate (3SP), a structural succinate analogue (21). The latter is thereafter activated to the corresponding 3SP-CoA thioester by a succinate-CoA ligase (SucCD) that is able to accept several structurally related substrates (22). So far, the metabolic fate of the sulfur moiety has been unclear.…”

“…Genotypic and phenotypic characterization of Tn5::mob-induced mutants as well as characterization of putatively involved enzymes helped to unravel the first steps of this pathway ( Fig. 2) (18,(20)(21)(22). Initial cleavage of DTDP by a disulfide reductase (LpdA) yields two molecules of 3-mercaptopropionic acid (3MP) (20).…”