Discovery and Characterization of Diazo-Forming Enzymes in the Cremeomycin Biosynthetic Pathway

Abstract

Nature constructs a wide variety of structurally complex and bioactive small molecules that have critical physiological and ecological roles. These natural products have served as an important source in developing therapeutics that treat a range of human conditions including cancer, infectious disease, and pain management. Investigating the biosynthesis of these natural products offers the opportunity to discover new bioactive small molecules as well as engineer these pathways for the production of novel compounds. Elucidating the enzymes that catalyze the chemical transformations within these biosynthetic pathways will enable applications in genome mining, biocatalysis, and synthetic biology that can afford therapeutic candidates. Here we describe the discovery and characterization of novel enzymes that install the reactive diazo functional group during cremeomycin biosynthesis. These efforts have provided insight into a long- standing challenge within the field of natural products chemistry and provide the knowledge for identifying and engineering novel diazo-containing small molecules. In Chapter 2 we describe our efforts to identify the biosynthetic gene clusters that are responsible for producing the diazo-containing natural products cremeomycin (cre) and lomaiviticin (lom). We undertook a genome mining strategy, based on the hypothesized intermediacy of the molecule 3-amino-4- hydroxybenzoic acid (3,4-AHBA) in cremeomycin biosynthesis and the polyketide nature of lomaiviticin, to identify the putative cre and lom gene clusters. These clusters were confirmed experimentally using heterologous expression and bacterial genetics, respectively. Based on the enzymatic chemistry encoded within the gene clusters we provided an updated biosynthetic hypothesis for cremeomycin and lomaiviticin assembly. In Chapter 3 we use a combination of in vivo and in vitro enzyme characterization, synthesis, and stable isotope feeding studies to elucidate the biosynthesis of cremeomycin up to the point of diazotization. We find that following 3,4-AHBA synthesis by the enzymes CreH and CreI, subsequent C2 hydroxylation and C4 O-methylation are carried out sequentially by the flavin-dependent monooxygenase CreL and the SAM-dependent O-methyltransferase CreN to afford 3-amino-2-hydroxy-4-methoxybenzoic acid (3,2,4- AHMBA). Thus, diazotization in cremeomycin biosynthesis occurs as the last step via late-stage N–N bond formation with the aniline 3,2,4-AHMBA. In Chapter 4 we identify and characterize a novel pair of enzymes that generate nitrite from L- aspartate. Feeding studies with 15N inorganic nitrogen salts suggest that nitrite is the source of the distal nitrogen atom in the diazo group of cremeomycin. In vitro biochemical reconstitution supports a model in which the flavin-dependent monooxygenase CreE catalyzes the six-electron N-oxidation of L-aspartate to nitrosuccinate with subsequent C–N bond cleavage by the aspartase homolog CreD to afford nitrite and fumarate. Interestingly, CreD and CreE appear to form a tightly regulated complex and this complex is essential for nitrite generation. Finally, in Chapter 5, we identify and conduct preliminary characterization of the diazo-forming enzyme from cremeomycin biosynthesis. Using a combination of an Escherichia coli-based heterologous expression system, lysate experiments, site-directed mutagenesis, and biochemical reconstitution with partially-purified enzyme, we demonstrate that the fatty acid-coenzyme A (CoA) ligase homolog CreM catalyzes diazotization of 3,2,4-AHMBA with nitrite to afford cremeomycin in vivo and in vitro. Our in vivo and in vitro investigations suggest that ATP is required for the diazotization reaction, which we propose is used to activate nitrite and potentially an N–N linked intermediate. Future efforts are focused on structural and further biochemical characterization of CreM in order to develop a detailed mechanistic understanding of the diazotization reaction including the role of ATP.