Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction ofC-nucleoside scaffolds

Abstract

24Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside 25 antibiotics, in which ribose and a pyrazole-derived base are linked by a C-glycosidic 26 bond, however, the logic underlying the biosynthesis of these molecules has 27 remained largely unexplored. Here, we report the discovery of the pathways for 28 FOR-A and PRF-A biosynthesis from diverse actinobacteria, and demonstrate that 29 their biosynthesis is initiated by a lysine N 6 -monooxygenase. Moreover, we show 30 that the forT and prfE (individually related to FOR-A and PRF-A biosynthesis) 31 mutants are correspondingly capable of accumulating the unexpected 32 pyrazole-related intermediates, compound 11 and 9a. We also decipher the 33 enzymatic basis of ForT/PrfE for the C-glycosidic bond formation in FOR-A/PRF-A 34 biosynthesis. To our knowledge, ForT/PrfE represents the first example of β-RFA-P 35 (β-ribofuranosyl-aminobenzene 5'-phosphate) synthase-like enzymes governing 36 C-nucleoside scaffold construction in natural product biosynthesis. These data 37 establish a foundation for combinatorial biosynthesis of related purine nucleoside 38 antibiotics, and also open the way for target-directed genome mining of 39

Synthetic and biosynthetic routes to nitrogen–nitrogen bonds

“…Their biosynthetic gene clusters were identified recently and both clusters contain spb38/spb40/ spb39 relevant genes forK/forJ/forL and pyrM/pyrN/pyrL (also called as prfK/prfJ/prfL) indicating similar hydrazineformation pathways. 81,85,86 Previous feeding studies of pyrazomycin 86 and formycin 87 have revealed nitrogen N1 in pyrazole from the N6 of L-lysine, consistent with the occurrence of pyrM and pyrN, and C1-4 carbons are derived from L-glutamate 87 (Fig. 17A).…”

Nitric oxide as a source for bacterial triazole biosynthesis

“…To connect two nitrogen atoms together, nature has devised different strategies. The most frequently used precursors for N-N bond formation are hydroxylamines (such as L-N 5 -OH-Ornthine in piperazate pathway 9 , N-isobutylhydrdoxylamine in valanimycin pathway 37 , and L-N 6 -OH-lysine in the pathways of s56-p1 14 , pyrazomycin 16 and formycin 17,38 ) and nitric acid, which derives from the α-amine of L-aspartic acid by the enzyme pair CreE and CreD 10 . The flavin monooxygenase CreE and nitrosuccinate lyase CreD were first characterized in the cremeomycin pathway, and their homologs were subsequently identified in the pathways of fosfazinomycin, and kinamycin, triacsins and alanosine 15,[21][22][23] .…”

Experimental and computational snapshots of C-C bond formation in a C-nucleoside synthase

“…Insights into the biosynthetic origins of bioactive C-nucleosides have resulted from the identification of the gene clusters encoding a number of these secondary metabolites [19][20][21][22][23][24][25][26][27][28]. In broad terms, the cellular pathways to these natural products use three distinct chemical strategies to make the key C-C bond (electronic supplementary material, figure S1).…”

“…Reattachment of the nucleobase to the ribose via a C-C bond then follows rotation of the base within the active site [26,27]. In the third strategy, formycin A and pyrazofurin A arise from direct coupling of a small heteroaromatic dicarboxylic acid to 5 0 -phosphoribose-1 0 -pyrophosphate (PRPP) with concomitant decarboxylation [22,30].…”