Biochemical characterization of a non-canonical branching module of the rhizoxin polyketide synthase

The biosynthesis of the -lactone moiety of the macrolide rhizoxin affords the incorporation of a rare β-branch by Michael addition of a C2 unit to the α,β-unsaturated polyketide intermediate in the branching module (KS-B-ACP). The branching module accepts amine- and carboxamide-substituted substrates to yield -lactam and glutarimide rings respectively. This result is relevant to the biosynthesis of glutarimide in cycloheximide-type of antibiotics. In the presence of methylmalonyl-CoA extender unit, the branching module forms a dual branched product. In a single step, two (α and β) branches are produced in a vinylogous stereoselective branching event. In addition to the formation of 6-membered rings, the branching module could also form an array of 5- to 10-membered lactones. The branching module is therefore an excellent candidate to generate non-natural and potentially bioactive compounds. The glutarimide moiety of the cycloheximide-type of antibiotics is also biosynthesized by a similar branching module (KS-X-ACP). However, the roles of the cryptic B and X domains remained unclear. By constructing chimeras of the B domain with the X domain and a homologous DH domain, this thesis reveals that the B/X domains do not play any catalytic role but are critical for maintaining the structural integrity of the KS-B didomain. Therefore, the KS domain is solely capable of the branching and cyclization reactions. Site-directed mutagenesis of the conserved amino acids of the branching-KS identified key residues for catalysis and sets the stage to probe further into the mechanistic details of the complex branching reaction. Taken together, the results from this thesis validate that the branching module exhibits tolerance towards a non-natural substrates to produce novel ring systems. It further provides a detailed functional analysis of its key components (KS and B)

Trapping of a polyketide synthase module after C−C bond formation reveals transient acyl carrier domain interactions

Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)‐bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter‐domain interactions after C−C bond formation in a chain‐branching module of the rhizoxin PKS. Mechanism‐based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C−C bond formation by cryo‐electron microscopy (cryo‐EM). The possible existence of two ACP binding sites, one of them a potential “parking position” for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C−C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines