Identification of the C‐Glycoside Synthases during Biosynthesis of the Pyrazole‐C‐Nucleosides Formycin and Pyrazofurin

C‐Nucleosides are characterized by a C−C rather than a C−N linkage between the heterocyclic base and the ribofuranose ring. While the biosynthesis of pseudouridine‐C‐nucleosides has been studied, less is known about the pyrazole‐C‐nucleosides such as the formycins and pyrazofurin. Herein, genome screening of Streptomyces candidus NRRL 3601 led to the discovery of the pyrazofurin biosynthetic gene cluster pyf. In vitro characterization of gene product PyfQ demonstrated that it is able to catalyze formation of the C‐glycoside carboxyhydroxypyrazole ribonucleotide (CHPR) from 4‐hydroxy‐1H‐pyrazole‐3,5‐dicarboxylic acid and phosphoribosyl pyrophosphate (PRPP). Similarly, ForT, the PyfQ homologue in the formycin pathway, can catalyze the coupling of 4‐amino‐1H‐pyrazole‐3,5‐dicarboxylic acid and PRPP to form carboxyaminopyrazole ribonucleotide. Finally, PyfP and PyfT are shown to catalyze amidation of CHPR to pyrazofurin 5′‐phosphate thereby establishing the latter stages of both pyrazofurin and formycin biosynthesis. Enzymatic C‐glycoside bond formation: The C‐glycoside synthases encoded in the formycin and pyrazofurin biosynthetic gene clusters are identified and compared by genomic analysis of the producing strains. Both enzymes catalyze decarboxylative C‐glycoside bond formation between phosphoribosyl pyrophosphate and a pyrazole nucleobase in reactions likely to involve electrophilic aromatic substitution.