Purification of heteromeric acetyl-CoA carboxylases from Escherichia coli for structure solution.

The primary role of acetyl-CoA carboxylases (ACCs) is to generate malonyl-CoA for use in fatty acid and lipid biosynthesis. However, malonyl-CoA is also used in other various metabolic processes such as secondary metabolite biosynthesis. The diverse utilization of malonyl-CoA makes ACCs targets for the development of inhibitors and also a target for engineering allosteric regulation for biofuel and secondary metabolite production. The ACC from Escherichia coli is representative most of bacterial systems, and is heteromeric, being comprised of four proteins encompassing three distinct subunits. Historically the purification of active E. coli ACC complexes has been problematic due to the reported facile dissociation into subunits. Most studies on heteromeric ACCs study the isolated subunits, which are active on their own. Nevertheless, in reconstituted systems, the subunits appear to have allosteric interactions. In this chapter, we provide methods to generate, purify and characterize these heteromeric ACCs complexes. We have used these methods to solve cryogenic electron microscopy structures of active E. coli ACC complexes. Purification of active ACC complexes represents a significant step forward in our ability to characterize how allosteric interactions and effectors alter catalytic activity. We expect future studies on the heteromeric ACC complexes will enable rational engineering of new antibiotics and biofuel production.