Ten equation. (iii) Utilization of CoA donors aside from succinyl-CoA. The assay mixture contained 0.two

June 30, 2023

Ten equation. (iii) Utilization of CoA donors aside from succinyl-CoA. The assay mixture contained 0.two mM DTNB, 10 mM 3SP, and an excess of AcdDPN7 in 50 mM Tris-HCl (pH 7.six)50 mM NaCl inside a final volume of 1 ml. Just 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 and other bacteria. lysR, transcription issue; act, acyl-CoA-transferase;acd, acyl-CoA dehydrogenase; ech, enoyl-CoA hydratase/KLF web isomerase; mdo, 3-mercaptopropionate dioxygenase; ahpd, alkylhydroperoxidase; bug, Bordetella uptake gene.nyl-CoA, maleyl-CoA, succinyl-CoA, itaconyl-CoA, glutaryl-CoA, and 3-thiaglutaryl-CoA. Soon after incubation for yet another minute, the reaction was started by addition of 42 g of purified recombinant ActTBEA6. The boost in absorbance was monitored at 412 nm. (iv) Utilization of CoA acceptors aside from 3SP. The assay mixture with a final volume of 1 ml in 50 mM Tris-HCl (pH 7.6)50 mM NaCl contained 0.1 mM succinyl-CoA, 10 g purified heterologous ActTBEA6, and five mM every of the following putative CoA acceptors: sodium acetate, sodium propionate, itaconic acid, sodium fumarate, mercaptosuccinic acid, or sodium glutarate. Stock solutions in the corresponding substrates had been adjusted to a pH range of 7.0 to eight.0 in advance. Just after 15 min of incubation at 30 , the reaction was stopped by addition of 30 l (15 [wt/vol]) trichloroacetic acid. Samples had been analyzed for formation on the corresponding CoA esters by HPLC-ESI-MS. Inactivation experiments. Hydroxylamine and sodium borohydride had been applied in two inactivation experiments. (i) Inactivation by hydroxylamine. A total of 210 g purified recombinant ActTBEA6 was incubated for ten min at 30 in 490 l 50 mM Tris-HCl (pH 7.six), with 150 mM NaCl, either CB2 Synonyms containing or lacking succinyl-CoA (2 mM). Subsequently, five l 1 M hydroxylamine option (in H2O [pH 7.0], adjusted with five M NaOH) was added to a final concentration of ten mM, and also the reaction mixture was incubated for an more ten min at 30 . Afterwards, the reaction mixture was diluted 1:ten with 50 mM Tris-HCl (pH 7.6)50 mM NaCl and stored on ice until enzyme activity was determined with the coupled spectrophotometric assay. Activity was measured in triplicate for the enzyme solutions incubated with or with out succinyl-CoA. (ii) Inactivation by sodium borohydride. A total of 210 g purified recombinant ActTBEA6 (from the very same batch pointed out above) was incubated for 10 min at 30 in 490 l 500 mM Tris-HCl (pH 7.6), either containing or lacking succinyl-CoA (two mM). Subsequently, five l 1 M sodium borohydride in 1 M NaOH was added, followed by addition of 5 l 1 M HCl quickly afterwards. The reaction mixture was incubated for an more 10 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 with all the coupled spectrophotometric assay. Activity was measured in triplicate for the enzyme solutions incubated with or without succinyl-CoA. Analysis of CoA ester formation by HPLC-ESI MS. Formation of 3SP-CoA in the course of enzyme assays was followed by high-pressure liquid chromatography in combination with electrospray ionization mass spectrometry (HPLC-ESI.