Fig. 1.

Mevalonate pathway and purification strategy. (A) Reaction scheme of the three enzymes involved in the mevalonate pathway. (B) Workflow applied in this work to identify and analyze the thiolase/HMGCS complex. Proteins in the cell extract were isolated by shotgun purification (SI Appendix). Two different crystalline forms appeared (green arrows) from a fraction. Crystals were washed and dissolved. The protein bands on SDS/PAGE of the dissolved crystals were used for mass spectrometric identification. Based on the protein-sequence analysis, the function of the crystallized proteins was predicted and the thiolase/HMGCS activity was measured by the HMGCR-coupled assay. The thiolase/HMGCS complex was purified again from the cell extract using the enzyme assay (target purification), and the purified enzyme complex was enzymologically characterized.