Oxygen-dependent enzymes involved in phosphonate biosynthesis

Microorganisms are an invaluable source of natural products. As humans, we have taken advantage of these small molecules to improve overall human health and quality of life. However, the rise in the number of pathogens that have become resistant to modern medicines requires action to deliver new compounds to treat disease. Natural products are a rich source of potential drug leads, as approximately 65% of our current antimicrobials are natural products or inspired by natural products. With the advent of genome sequencing, we now have the ability to quickly identify new biosynthetic gene clusters with the capacity to produce new natural products and quickly identify the genes required for the production of a natural product. Characterization of these biosynthetic gene clusters allow us to identify the enzymes required for the biosynthesis and functionalization of these compounds and the enzymes that may be used for inactivation of toxic compounds. They also inform us on strategies that can be used to manipulate these organisms to produce derivatives of potentially useful natural products. Among the most useful natural products are the phosphonic acids, which contain a carbon-phosphorus bond. Over the years, these small molecules have shown great success as drugs and herbicidal compounds, thus it is prudent to facilitate the rapid discovery and characterization of new phosphonate natural products. The work in this thesis focuses on the characterization of the enzymes needed for formation and functionalization of many different phosphonic acid biosynthetic pathways. Non-heme iron-dependent oxygenases are important enzymes in all domains of life that catalyze various chemical reactions involving the functionalization of an un-activated carbon atom. These enzymes are present in many phosphonate biosynthetic pathways. Chapter 2 will focus on the in vitro biochemical characterization of the TauD-family dioxygenases that are involved in the biosynthesis of dehydrophos, a compound with proven antibiotic activity, fosfazinomycin, a compound containing a unique phosphono-hydrazide linkage, and hydroxyphosphonocystoximate, a phosphonate containing an unusual thiohydroximate moiety. Here, I show that these enzymes share little identity with one another, which drive the substrate specificity of these oxygenases. Oxime-containing natural products are uncommon in nature, with even less being understood about how organisms make these functional groups. Phosphonocystoximic acid and its hydroxylated congener, hydroxyphosphonocystoximic, are two phosphonate natural products containing a free oxime moiety. Chapter 3 will focus on the enzymes required for N-oxidation and oxime formation during the biosynthesis of these two natural products. We have identified an NADPH- and FAD-dependent enzyme responsible oxidation of a primary amine to the oxime that is catalyzed early in the biosynthesis of these natural products. Phosphonothrixin is a potent herbicidal natural product that is produced by Saccharothrix sp. ST-888. Until recently, the biosynthetic gene cluster responsible for production of this compound remained elusive. I will describe in vitro characterization of three enzymes encoded within the phosphonothrixin biosynthetic gene cluster in Chapter 4. Additionally, the isolation of dihydroxypropylphosphonic acid from culture extracts of Saccharothrix sp. ST-888 have allowed us to formulate a hypothesis for the biosynthesis of the herbicidal compound