Characterization of Catalytic Diversity in Megaenzyme Systems

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. Bacteria and fungi produce a wide range of secondary metabolites. These natural chemicals give microbes growth advantages or kill competing organisms. The biological activities of these compounds also make them useful to society as agricultural agents, pesticides and "green" chemicals. Many of the proteins responsible for synthesis of these secondary metabolites constitute large, complex enzyme systems (megaenzymes or megasynthases) called polyketide synthases and nonribosomal peptide synthetases. Canonical megasynthases are formed of repeating combinations of specific domains, and produce a variety of small molecule compounds from monomer building blocks. Intriguingly, in some systems, the diversity is further increased by co-opting proteins, protein domains, and/or substrates from various other cellular processes. The goal of this research program is to investigate and structurally characterize megaenzyme systems that display atypical composition and mechanisms resulting from the inclusion of these novel components. The first two unusual megaenzyme systems we will investigate are andrimid synthase and the depsipeptide synthases. Andrimid synthase is a megaenzyme that has co-opted a transglutaminase homologue, AdmF, into its synthetic cycle. Transglutaminases are usually associated with blood clotting and protein crosslinking, but AdmF is a free-standing protein that catalyzes the joining of a fatty acid and an amino acid which are bound to megaenzyme carrier proteins. Depsipeptides, such as cereulide and antimycin, are small molecules with amide and ester linkages between monomers. Depsipeptide synthases use a-keto acids as substrates and reduce them in a co-opted ketoreductase domain to produce a-hydroxy acids to incorporate into their product. We will study the structure and function of noncanonical megaenzyme systems using X-ray crystallography, mutational and kinetic analysis, and other biochemical and biophysical approaches. This will provide fundamental understanding of the mechanisms employed to make diverse chemical products. These mechanisms could possibly be exploited to produce novel biochemicals for use in industry and agriculture.