Elucidation of the N-Nitroglycine Biosynthetic Pathway: Towards the Discovery of Novel Natural Product Nitramines

Benjamin Rathman, Gabriel Padilla, Jonathan Caranto

University of Central Florida

N-Nitroglycine (NNG) is a bacterial metabolite that features an unusual α-nitramino functional group. Herein, we report the discovery of the NNG biosynthetic gene cluster from Streptomyces noursei. AntiSMASH prediction of biosynthetic gene clusters within the S. noursei genome revealed homologs of hydrazinoacetic acid (HAA)-producing enzymes reported in the literature; therefore, one proposed pathway was that HAA is a precursor for NNG biosynthesis. This hypothesis was supported by ¹⁵N-isotope feeding and in vitro reconstitution experiments. The AntiSMASH analysis further suggested that NNG is a precursor for a more complex natural product. To enable detection of NNG-derived natural products, we developed a methodology to quantify nitramines via alkaline alkylation, which enables nitrite elimination that can be detected and quantified by a Griess assay. We leverage the unique reactivity of nitramines to guide the fractionation of bacterial matrices, allowing for the identification of nitramine containing natural products.