Authors: Kwo-Kwang Abraham Wang
Source: IDEALS (University of Illinois Urbana-Champaign) (article, 2018-12-19)
Field: Chemistry — Organic Chemistry

Investigating the biosynthesis of fosfazinomycin Phosphonic and phosphinic acid natural products are characterized by their carbon-phosphorus bonds, and exhibit a variety of bioactivities. Further, these compounds often contain unusual structural features, which arise from remarkable enzymatic transformations. One example comes from fosfazinomycin, an antifungal compound bearing a unique phosphonohydrazide (C-P-N-N-C) linkage that joins a valine-arginine dipeptide to methyl-2-hydroxyphosphonoacetate (Me-HPnA). This thesis describes my efforts towards determining how the nitrogen-nitrogen (N-N) bond is crafted and installed during fosfazinomycin biosynthesis. \n\nThe building blocks used to construct natural products are often derived from metabolites coming from central metabolism. Chapter 2 addresses my efforts to ascertain the biosynthetic origins of the N-N bond in fosfazinomycin. I was able to optimize conditions for the production of fosfazinomycin from its native Streptomyces producing organism and conducted stable isotope labeling experiments. The results of those studies revealed that the primary amine from aspartic acid is incorporated into the phosphonohydrazine moiety of fosfazinomycin. Further purification and spectroscopic efforts additionally demonstrated that the nitrogen atom originating from aspartic acid is donated to the site proximal to the phosphorus. \n\nInterestingly, a number of enzymes are conserved in the pathways to fosfazinomycin and to kinamycin, another natural product that contains an N-N bond but is otherwise structurally unrelated. In Chapter 3, I describe the discovery of a recurring modular pathway for the installation of hydrazine onto diverse chemical structures. Using a combination of isotope labeling studies and in vitro enzymatic assays, I illustrate a biosynthetic route wherein nitrous acid, liberated from aspartic acid, is used to build a hydrazine synthon that, after a number of steps, is eventually condensed onto the side chain of glutamic acid to make glutamylhydrazine. Glutamylhydrazine then serves as common carrier molecule for hydrazine, which can subsequently be deposited onto varying chemical scaffolds. \n\nPrevious work had shown that fosfazinomycin biosynthesis proceeds in a convergent manner with the Me-HPnA and argininylmethylhydrazine being formed independently. My attempts at reconstituting the transfer of hydrazine from glutamylhydrazine onto the carboxyl group of arginine are described in Chapter 4. FzmA, an asparagine synthetase homolog, is a prime candidate for effecting this transformation, and I used a number of in vitro enzymatic assays to test for the desired FzmA activity. Unfortunately, I was unable to reconstitute the formation of argininylhydrazine using FzmA. I also highlight the discovery of a PqqD-like enzyme and a nucleotidyltransferase homolog that may be involved in fosfazinomycin biosynthesis. \n\nHow the N-N bond is crafted in fosfazinomycin still remains a mystery. Chapter 5 recounts my failed efforts towards reconstituting the enzymatic formation of the N-N bond. I discuss a number of potential biosynthetic strategies that may be at play in N-N bond construction, and the biochemical approaches I took to investigate them. I also describe the identification of FzmP as a heme protein that is potentially involved in N-N bond formation. Biosynthesis ; Chemistry ; Biochemistry ; Gene Synthesis and Catalytic Reactions