Abstract 1279 De novo biosynthesis of azide by a promiscuous N-nitrosylase

Azides are energy-rich compounds endowed with diverse representation in broad scientific disciplines, including material science, synthetic chemistry, pharmaceutical science, and chemical biology. Notably, the azide-alkyne cycloaddition reaction has revolutionized the field of chemical biology due to its "bio-orthogonal" and "clickable" features. The broad application of azide in chemistry has raised significant interest in the azidation reaction using enzymes. Specifically, multiple non-heme iron halogenases and oxygenases have been engineered to catalyze azide transfer, typically from an inorganic azido donor to a few selected substrates. Alternatively, the azide group might be directly biosynthesized in situ without feeding the bio-orthogonal moiety itself. This new approach would enable the biogenesis of azide under mild, physiological conditions, allowing for tunable production of this bio-orthogonal handle for in vivo labeling with low metabolic background. However, enzymes for de novo azide biosynthesis remain largely unknown. We recently discovered the first natural enzyme, a promiscuous ATP and nitrite-dependent enzyme, which catalyzes organic azide synthesis through sequential N-nitrosation and dehydration of aryl hydrazines. Through biochemical, structural, and computational analyses, we further proposed a plausible molecular mechanism for azide biosynthesis that sets the stage for future biocatalytic applications and biosynthetic pathway engineering. This research was financially supported by a grant to W.Z. from the NIH (R01GM136758).