Pyrazofurin Biosynthesis Involves Nonenzymatic Ring Contraction of a Pyridazine Intermediate Triggered by a Rieske Enzyme-Catalyzed Oxygenation

Pyrazofurin and formycin A are two pyrazole-containing <i>C</i>-nucleosides, exhibiting antibacterial, antiviral, and antitumor activities. Despite previous studies elucidating the early biosynthetic steps of pyrazole formation, how the linear hydrazone intermediate is cyclized to a pyrazole ring remains unknown. Herein, substrate analogs are used to show that the amidohydrolase PyfA mediates intramolecular cyclization of 2-hydrazinylidenepentanedioic acid to yield a 4,5-dihydropyridazine ring, which can undergo PyfO-catalyzed dehydrogenation to generate a pyridazine product. More importantly, the Rieske enzyme PyfB is demonstrated to catalyze oxygenation of 4,6-dihydroxypyridazine-3-carboxylic acid. The resulting six-membered pyridazine product is found to undergo uncatalyzed rearrangement to the five-membered pyrazole ring. This work thus highlights a unique pyrazole-forming pathway involving a rare pyridazine intermediate that undergoes oxidation-triggered nonenzymatic ring contraction to yield the pyrazole core.