Biosynthesis and Cellular Actions of Bioactive Natural Products

The utilization of natural products as therapeutic agents has been an invaluable resource throughout medicinal history. Through the combined application of combinatorial biosynthesis, the modification of a natural product by engineered enzymes, and the isolation of new bioactive natural products, the discovery of new therapeutic agents may be achieved. This work shows the novel enzymatic interaction in the biosynthesis of a known therapeutic agent and the isolation of a new bioactive natural product. Azinomycin A and B are antitumor natural products, isolated from Streptomyces sahachiroi. It was proposed that the biosynthesis of azinomycins is achieved through polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) machinery. In order to characterize the role of the PKS (AziB), enzymatic assays were analyzed by HPLC, LC-MS, and spectrophotometric methods. Although AziB was predicted to catalyze the production of 5-methyl-1-napthoic acid, a building block of azinomycins, a thioesterase (AziG) was found to be essential. Kinetic and crystallographic studies suggested the importance of the interaction of AziB and AziG to facilitate optimal enzymatic activity. The derivative of the AziB-AziG product is hypothesized to be assembled into the azinomycin backbone by NRPSs. In order to confirm the significance of a NRPS (AziA2) in the azinomycin biosynthetic pathway, gene knockout studies were performed. Fermentation of ΔaziA2 S. sahachiroi led to an cryptic overproduction of dimethyl furan-2,4-dicarboxylate. This showed an example of bacterial adaptation where bacteria start overproducing a new secondary metabolite to deal with the absence of a bioactive natural product. A novel bioactive 40-membered macrolactone, Nuiapolide, was identified from a cyanobacterium collected from the Hawaiian ocean. This molecule has a rare tert-butyl side chain and nine hydroxyl groups distributed through the large hydrocarbon ring structure. Nuiapolide could inhibit metastatic activity of Jurkat at the concentration of as low as 1.3 μM without killing the cells. In this dissertation, the new functionality of a class of enzyme, the effect of biosynthetic disruption, and a novel anti-metastatic natural product from a marine source will be discussed. This information leads to a new understanding of natural products and their biosynthesis, which will eventually help researchers developing novel therapeutic agents