Ten equation. (iii) Utilization of CoA donors aside from succinyl-CoA. The assay mixture contained 0.2 mM DTNB, 10 mM 3SP, and an excess of AcdDPN7 in 50 mM Tris-HCl (pH 7.6)50 mM NaCl in a final volume of 1 ml. Soon after preincubation for 1.5 min at 30 , one of several following CoA esters was added to a final concentration of 0.13 mM: acetyl-CoA, propionylCoA, butyryl-CoA, valeryl-CoA, isobutyryl-CoA, isovaleryl-CoA, croto-jb.asm.orgJournal of BacteriologySuccinyl-CoA:3-Sulfinopropionate CoA-TransferaseFIG two Gene organization in proximity of act orthologues in V. paradoxus strain TBEA6 along with other bacteria. lysR, transcription element; act, acyl-CoA-transferase;acd, acyl-CoA dehydrogenase; ech, enoyl-CoA hydratase/isomerase; mdo, 3-mercaptopropionate dioxygenase; ahpd, alkylhydroperoxidase; bug, Bordetella uptake gene.nyl-CoA, Factor Xa Inhibitor web maleyl-CoA, succinyl-CoA, itaconyl-CoA, glutaryl-CoA, and 3-thiaglutaryl-CoA. After incubation for one more minute, the reaction was started by addition of 42 g of purified recombinant ActTBEA6. The raise in absorbance was monitored at 412 nm. (iv) Utilization of CoA acceptors besides 3SP. The assay mixture having a final volume of 1 ml in 50 mM Tris-HCl (pH 7.six)50 mM NaCl contained 0.1 mM succinyl-CoA, ten g purified heterologous ActTBEA6, and five mM every single of the following putative CoA acceptors: sodium acetate, sodium propionate, itaconic acid, sodium fumarate, mercaptosuccinic acid, or sodium glutarate. Stock options with the corresponding substrates had been adjusted to a pH range of 7.0 to eight.0 in advance. Right after 15 min of incubation at 30 , the reaction was stopped by addition of 30 l (15 [wt/vol]) trichloroacetic acid. Samples have been analyzed for formation in the corresponding CoA esters by HPLC-ESI-MS. Inactivation experiments. Hydroxylamine and sodium borohydride have been applied in two inactivation experiments. (i) Inactivation by hydroxylamine. A total of 210 g purified recombinant ActTBEA6 was incubated for 10 min at 30 in 490 l 50 mM Tris-HCl (pH 7.6), with 150 mM NaCl, either containing or lacking succinyl-CoA (two mM). Subsequently, five l 1 M hydroxylamine resolution (in H2O [pH 7.0], adjusted with five M NaOH) was added to a final concentration of ten mM, plus the reaction mixture was incubated for an extra ten min at 30 . Afterwards, the reaction mixture was diluted 1:10 with 50 mM Tris-HCl (pH 7.6)50 mM NaCl and stored on ice until enzyme activity was determined using the coupled spectrophotometric assay. Activity was measured in triplicate for the enzyme options incubated with or devoid of succinyl-CoA. (ii) Inactivation by sodium borohydride. A total of 210 g purified recombinant ActTBEA6 (from the exact same batch IL-2 web talked about above) was incubated for ten min at 30 in 490 l 500 mM Tris-HCl (pH 7.six), either containing or lacking succinyl-CoA (2 mM). Subsequently, 5 l 1 M sodium borohydride in 1 M NaOH was added, followed by addition of five l 1 M HCl immediately afterwards. The reaction mixture was incubated for an further 10 min at 30 . Afterwards, the reaction mixture was diluted 1:10 with 50 mM Tris-HCl (pH 7.six)50 mM NaCl and stored on ice till enzyme activity was determined with the coupled spectrophotometric assay. Activity was measured in triplicate for the enzyme options incubated with or without succinyl-CoA. Evaluation of CoA ester formation by HPLC-ESI MS. Formation of 3SP-CoA for the duration of enzyme assays was followed by high-pressure liquid chromatography in mixture with electrospray ionization mass spectrometry (HPLC-ESI.