Discovery and Characterization of the Streptozotocin Biosynthetic Pathway

Abstract

Natural products have historically provided a rich source of medicines, and natural product isolation and characterization continue to drive drug discovery and design. As the availability of genomic DNA sequences in public databases continues to increase due to decreasing costs, we now have access to an abundance of gene clusters that encode uncharacterized natural product biosynthetic pathways. Therefore, by understanding the biosynthetic processes that generate natural products, we will advance our ability to mine this abundance of available microbial sequencing data and may facilitate the discovery of additional naturally-derived bioactive compounds. Detailed in vitro characterization of enzymes that catalyze the chemical transformations within these biosynthetic pathways will enable applications in genome mining, biocatalysis, and design of organometallic catalysts. Here we describe the discovery and characterization of novel enzymes that install a reactive N-nitroso functional group during streptozotocin biosynthesis. These efforts have provided our first insights into enzymatic N-nitrosation in biological systems. With the elucidation of the genetic and biochemical basis for N-nitroso biosynthesis, our work now enables investigations of the biosynthesis of additional N-nitroso and N-N bond containing natural products and targeted genome mining efforts to identify additional N-nitroso compounds. In Chapter 2 of this dissertation, we present the identification and validation of the putative streptozotocin biosynthetic gene cluster. This gene cluster was identified using a resistance-guided genome mining strategy. Experimental work linked this gene cluster to the production of streptozotocin. in vivo genetic analysis revealed four essential biosynthetic genes. In vivo feeding experiments and in vitro biochemical characterizations also outlined the early stages of streptozotocin biosynthesis. Chapter 3 of this work details the biochemical characterization of the N-nitrosating metalloenzyme SznF. Biochemical characterization revealed a sequential oxygenation reaction on a modified L-arginine substrate. Reaction intermediates were accessed via chemical synthesis, and stable isotope tracer experiments provided insights into the mechanism of N-nitroso formation. We also pursued structural characterization of SznF and used site-directed mutagenesis experiments to reveal two distinct metallocofactor sites in this enzyme that have distinct catalytic capabilities. Future efforts are focused on further structural and spectroscopic characterization to elucidate the mechanism of SznF-mediated N-oxidation and N-N bond formation. Finally, in Chapter 4, we will describe the roles of the ATP-grasp enzymes encoded in the streptozotocin biosynthetic gene cluster and assess the distribution of SznF-homologs in microbial genomes. We have reconstituted the in vitro activity of SznH and K and established they are L-amino acid ligases. We have concluded that these two enzymes further modify an N-nitrosourea-containing amino acid to generate a tripeptide product. These results suggested that additional enzyme(s) may be required for completing streptozotocin biosynthesis. Finally, phylogenetic analysis of SznF and SznF-neighborhood revealed a widely-distributed enzyme class in multiple genus of bacteria in different environments. Future efforts are focused on identifying additional enzymes required to complete streptozotocin biosynthesis and biochemically characterizing SznF-homologs identified in our bioinformatic analyses.