C16 coa

The oxidation rate of C16-CoA was measured as reported previously [15 (link)]. C16-CoA, was purchased from Sigma (Steinheim, Germany). Reaction products were detected by LC-ESI-MS/MS in positive ionization mode according to the published literature [15 (link)].

10 μM of Rv0158 protein (in 50 mM Tris pH 7.5, 100 mM NaCl) was labelled using RED-NHS MonolithTM Protein Labeling Kit (NanoTemper Technologies, cat. no.- MO-L011) according to manufacturer’s instructions. Each of the small molecule ligands (coA (Sigma Aldrich, cat. no.- C4282), acetyl-CoA (Sigma Aldrich, cat. no.- A2056), propionyl-CoA (Sigma Aldrich, cat. no.- P5397), malonyl-CoA (Sigma Aldrich, cat. no.- M4263), methylmalonyl- CoA (Sigma Aldrich, cat. no.- M1762), C10-CoA (Sigma Aldrich, cat. no.- D5269), C12-CoA (Sigma Aldrich, cat. no.- L2659), C16-CoA (Sigma Aldrich, cat. no.- P9716), C18-CoA (Sigma Aldrich, cat. no.- S0802)) were titrated against the labeled protein in 1:1 dilution series starting with the highest ligand concentration of of 75 μM. A total of 16 twofold serial dilutions of the target ligand were prepared. The labeled protein was added such that the final concentration of the protein is 5 nM. Experiments were carried out using MonolithTM NT.115 MST Standard Capillaries (NanoTemper Technologies, cat. no.- MO-K022) and measured using a Monolith NT.115 instrument with MO.Control software. Curves were fitted with a single-site binding model using PALMIST 1.5.6 software (Scheuermann et al., 2016 (link)) and figures were generated using GUSSI software v1.1.0 (Brautigam, 2015 (link)).