Bacterial, fungal, and plant secondary metabolic pathways afford a huge library of elaborate organic molecules known as natural products. Polyketides and nonribosomal peptides are natural products that are biosynthesized from multienzyme complexes known as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), respectively. PKSs and NRPs utilize simple acyl-CoA or amino acid building blocks for the assembly of structurally diverse natural products. Because both polyketides and nonribosmal peptides exhibit a wide range of biological activities, they have been utilized as therapeutics and are important for drug discovery. The objective of this dissertation is to improve the structural understanding of how the biosynthetic machinery associated with polyketide and nonribosomal peptide biosynthesis generates such a large array of complex natural products.
In type II PKS biosynthesis, aromatase/cyclases (ARO/CYCs) are responsible for the generation of set C7-C12 or C9-C14 ring patterns through the regiospecific cyclization of reactive poly-beta-ketone intermediates. In Chapter 2, the first co-crystal structures of WhIE bound to four distinct pantetheine linked polyketide chain mimics are discussed. This work provides the first snapshot of ARO/CYC binding modes which, until now, have remained hypothetical. Further, this is the first successful application of polyketide mimics to investigate the structures and functions of ARO/CYCs.
Methyl-, ethyl-, and malonyl-CoA extender units are the most common building blocks employed by type I modular PKSs for natural product biogenesis. However, certain type I modular PKSs incorporate non-canonical long chain extender units to augment the structural diversity of a polyketide scaffold. Typically, crotonyl-CoA carboxylase/reductases (CCRs) are responsible for the generation of extravagant PKS extender units, whereas acyl-CoA carboxylases (ACCases) are responsible for the production of canonical short chain extender units. In Chapter 3, we discuss the structure and function of a novel ACCase β- subunit, MccB from the stambomycin biosynthetic pathway that is capable of generating long chain extender units. The crystal structures of apo and hexanoyl-CoA bound MccB were solved, providing a structural basis for unusual ACCase-mediate extender formation during stambomycin biogenesis.
Typically, in type I modular PKS biosynthesis, thioesterases (TEs) are responsible for product hydrolysis from the enzymatic assembly line. In some PKS/NRPS hybrid systems, however, a TE domain is absent and instead a terminal condensation domain (C domain) is required for product liberation from the enzymatic assembly line. The enacyloxin biosynthetic pathway is an example of a PKS/NRPS hybrid that utilizes a standalone NRPS condensation domain, bamb_5915, for product release through an intermolecular transesterification reaction between shikimate and a mature polyketide chain. The crystal structure and functional analysis of bamb_5915 is described in Chapter 4. In addition, bioinformatic analysis of PCP_5917 and other NRPS/PKS hybrid pathways is discussed. The information gleaned from these studies will aid in the generation of “mixed” PKS/NRPS natural products.
Genome mining of environmental DNA is an emerging method that has led to the identification of novel PKS natural product pathways. The arixanthomycins are a group of recently discovered natural products that were identified through the heterologous expression of environmental DNA derived gene clusters in Streptomyces. Chapter 5 reports the crystal structure of ARX 21, a novel C-17 and C-19 reducing ketoreductase, and in vitro reconstitution of an unusual C9-C14 reducing ARO/CYC from the arixanthomycin pathway.