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

Abstract

Formycin A and pyrazofurin are two naturally occurring pyrazole-derived C-nucleosides with antibacterial and antiviral activities. While earlier studies have established the chemistry of C-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 N⁶-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, N-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 N-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.

Figure 1.

(A) Structures of formycin A (1) and pyrazofurin (2). The pyrazole rings are highlighted in green and the C-glycosidic bond is marked in red. (B) Reaction schemes of C-glycosidation catalyzed by ForT and PyfQ.

Figure 2.

(A) The for and pyf biosynthetic gene clusters. Homologous genes that are likely involved in pyrazole ring formation are colored. Genes with a light blue background in the table are proposed to be involved in the final steps of pyrazole formation. (B) Formation of hydrazinoacetic acid (11) in the biosynthesis of s56-p1 (8). (C) Reported PyfG-catalyzed coupling of l-Glu and 9 to form l-13. While the activity of ForJ was not tested in the previous report, ForJ is expected to catalyze the same reaction as PyfG. The proposed formation of l-14 from l-13 is also shown here. (D) Reaction catalyzed by saccharopine dehydrogenase. (E) Previously demonstrated ForJ-catalyzed hydrazine formation in valanimycin biosynthesis using ester 17 as the substrate which is a mimic to the proposed ester intermediate involved in the ForJ/PyfG reaction.^,

Figure 3.

Proposed intramolecular cyclization catalyzed by ForM/PyfJ and the subsequent amide bond hydrolysis catalyzed by ForQ/PyfA.

Figure 4.

N-Acylation and subsequent amide bond hydrolysis in the biosynthesis of (A) triacsin A (24) and (B) actinopyridazinone A (25).

Figure 5.

(A) Reaction catalyzed by glycinamide ribonucleotide synthetase (GARS). (B) Reactions catalyzed by ForM/PyfJ and ForR/PyfK using l-14 or d-14 as substrates. LCMS analysis of the ForM and the ForM-ForR coupled reactions using (C) l-14 or (D) d-14 as substrates.

Figure 6.

(A) Reactions catalyzed by ForJ/PyfG and ForL. LCMS analysis of the (B) ForJ and the (C) ForJ-ForL coupled reactions.

Figure 7.

Proposed pathways of pyrazole ring formation in the biosynthesis of formycin A and pyrazofurin. AA indicates glycine, l-serine, l-alanine, l-threonine, or l-proline. The black solid arrows denote the confirmed steps in this study, while the dashed arrows indicate the proposed late steps.