Higher-order assemblies of biosynthetic multienzymes

This work provides a biochemical and structural analysis of two giant enzyme systems relevant for human health. Most enzymes have a single active site and turnover freely diffusing substrates. However, for biosynthetic pathways involving unstable intermediates, multienzymes have evolved that carry multiple enzymatic sites for subsequent reaction steps and use carrier proteins to shuttle covalently tethered substrates between active sites. Two prominent multienzyme systems are bacterial modular polyketide synthases (modPKSs) and human acetyl-CoA carboxylases (ACC). ACC catalyses the carboxylation of acetyl-CoA to malonyl-CoA, which serves as a substrate for fatty acid biosynthesis by fatty acid synthase. ModPKS are prokaryotic proteins that share a common evolutionary ancestor with FAS. They use malonyl-CoA and other acyl-CoA building blocks to generate highly complex bioactive natural products, the polyketides, with great potential as drug candidates.
Chapters 2 and 3, respectively, report the in vivo and in vitro analysis of the mupirocin PKS from Pseudomonas fluorescens. For in vivo analysis, I established methods for knocking-out, mutating and fluorescent protein labelling of mupirocin PKS proteins and analysed mupirocin production as a functional read-out. The results demonstrate that different labelled mupirocin PKS proteins localize to the cell pole and maintain product formation, while the determinants for localization remain to be defined. To further analyse protein interactions involved in this process, we aimed at assembling the entire ~1.2 MDa mupirocin PKS from individual proteins recombinantly expressed in insect cells. Due to challenges cloning large and repetitive genes, full reconstruction of the entire mupirocin PKS was not achieved. Biophysical and structural analysis by cryo-electron microscopy revealed that isolated mupirocin PKS proteins are predominantly homodimeric and lack higher order assemblies. The methods and materials derived here still provide an important stepping stone towards in vivo studies of modPKS assembly dynamics combined with in vitro reconstitution of an entire modPKS.
In chapter 4, I analyse the assembly of recombinant human ACC into active filaments triggered by its principal activator citrate. It has long been known to cause filament formation of the otherwise dimeric ACC, but the mechanistic basis for citrate action remained unknown. Here, we combine structural analysis of ACC filaments by cryo-EM and of ACC fragments by crystallography with biophysical and biochemical characterization of mutants. We demonstrate in atomic detail how citrate binds at the interface of two non-catalytic domains to ACC, aligning domains forming an active state that is conformationally locked by filament formation. Overall, we solve a 60-year puzzle of regulation of a key human enzyme and provide new paradigms for enzyme regulation and citrate sensing.