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UBC Theses and Dissertations

Building chemical tools from the indolmycin biosynthetic pathway Hoffarth, Elesha Rene


Natural products are essential to the discovery of new drugs, including antibiotics. Industrial interest in natural products has declined since the 1980s, but advances in biocatalysis and biosynthetic knowledge have helped revive interest in natural products as these advances contribute to more feasible discovery, production and derivatization of natural products. To continue this industrial interest in natural products and their related compounds, work should be done to accumulate more biosynthetic knowledge to further improve methods of discovery, production and derivatization and facilitate more widespread use of biocatalysts. Indolmycin is a natural product with antibiotic activities against methicillin-resistant Staphylococcus aureus, Helicobacter pylori and Plasmodium falciparum, whose biosynthetic pathway is shown here to be a source of new biochemical tools. First, in order to better understand the unique reactivity of the rare oxygen- and pyridoxal 5′-phosphate (PLP)-dependent arginine desaturases discovered from the indolmycin biosynthetic pathway, the first X-ray crystal structure of an arginine desaturase was solved. This structure showed an active site that was highly similar to the related oxygen- and PLP-dependent hydroxylases. Catalytic residues for the arginine desaturases were uncovered by creating mutagenic variants based on the crystal structure information. Second, sequence similarity analysis and side-product analysis were done, which further supported a higher similarity to the arginine hydroxylases than was originally predicted. Additionally, superoxide was shown to be an intermediate of the arginine oxidase mechanism for the first time through EPR and cytochrome c assays. Based on this information, a unified mechanistic hypothesis is proposed which suggests that desaturation and hydroxylation may be differentiated by the presence/position of water in the active site. Third, the indolmycin biosynthetic enzymes are used in conjunction with a promiscuous tryptophan synthase and a three-step chemical synthesis to produce indolmycin and several novel halogenated derivatives. Derivatives with fluorinated indole substitutions showed a moderate bioactivity against S. aureus and could be useful in developing indolmycin for clinical use. Overall, this work uses the indolmycin biosynthetic enzymes to expand the known biocatalytic repertoire with the hope that it can contribute to more widespread use of biocatalysts in the production of natural product-derived molecules.

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