Enzymatic Activity Assay of the Thiolase/HMGCS Complex.

The assay coupled with HMGCR was carried out with a Cary-4000 UV-visible spectrometer (Agilent) at 60 °C by following NADPH consumption at 340 nm using 10-mm quartz cuvettes (Hellma). To measure the activity of the fractions obtained in the purification steps, the assay solution contained 100 mM Hepes-KOH pH 7.5, 2 mM acetyl-CoA, 450 µM NADPH, and 2 µM HMGCR. Kinetic parameters of the purified thiolase/HMGCS complex were determined at 60 °C using the assay solution containing 500 mM potassium phosphate buffer pH 7.5, 400 µM NADPH, 2 µM HMGCR, 4.5 µg/mL thiolase/HMGCS complex, and varying concentrations of acetyl-CoA. The liquid chromatography (LC)-MS experiments were done with the Agilent 6550 iFunnel Q-TOF LC/MS system equipped with an electrospray ionization source set to positive ionization mode. Compounds were separated on an RP-18 column (50 × 2.1 mm, particle size 1.7 µm, Kinetex XB-C18; Phenomenex) using a mobile phase system composed of 50 mM ammonium formate pH 8.1 and methanol. Assays contained 100 mM Hepes-KOH pH 7.5, 0.1 mg/mL thiolase/HMGCS complex, and 2 mM acetyl-CoA, and, in the coupled assay, additionally contained 200 µM NADPH and 1.35 µM HMGCR. Samples were quenched with 5% formic acid (final concentration) and centrifuged for 5 min at 17,000 × g to remove precipitated proteins, and then the supernatant was analyzed by LC-MS. For this assay, the thiolase/HMGCS complex stored for about 2 wk at −80 °C was used. The activity of the stored preparation was 15% of that of the fresh sample. Compounds in the assay were quantified using external standard curves prepared in the same buffer system (SI Appendix, Fig. S13).