Revision of the Formycin A and Pyrazofurin Biosynthetic Pathways Reveals Specificity for <scp>d</scp>-Glutamic Acid and a Cryptic <i>N</i>-Acylation Step During Pyrazole Core Formation

Formycin A and pyrazofurin are two naturally occurring pyrazole-derived <i>C</i>-nucleosides with antibacterial and antiviral activities. While earlier studies have established the chemistry of <i>C</i>-glycosidic bond formation as well as the subsequent steps in the biosynthesis of formycin A and pyrazofurin, how the pyrazole ring itself is constructed remains elusive. While N-N bond formation in the pyrazole ring was previously reported to involve coupling of <i>N</i>⁶-hydroxylated l-lysine and l-glutamic acid catalyzed by the hydrazine synthetase PyfG, herein PyfG and its homologue ForJ are shown instead to recognize d-glutamate instead of l-glutamate. The hydrazine product of ForJ/PyfG catalysis then releases α-hydrazino d-glutamic acid upon processing by the NAD-dependent oxidoreductase ForL. Furthermore, <i>N</i>-acylation of α-hydrazino d-glutamate with an amino acid catalyzed by the ATP-grasp ligase ForM/PyfJ is indispensable for recognition by the FAD-dependent oxidoreductase ForR/PyfK to perform dehydrogenation of the Cα-N bond and thereby form a hydrazone intermediate. This work not only demonstrates that d-glutamic acid is the correct substrate for hydrazine biosynthesis but also reveals a cryptic <i>N</i>-acylation step in the assembly of the pyrazole core. These results thus provide significant insights into the biosynthesis of pyrazole rings that are rarely seen in natural products.