Meroterpenoids are known for their distinct structure and hybrid biosynthetic origin. The biosynthetic gene clusters (BGCs) of several well-characterized meroterpenoids contain three genes whose functions have remained elusive. Recent studies on nonmeroterpenoid pathways suggest that these genes may be involved in nitrite-dependent N-N bond formation. In this study, we show that one of these genes, fur5, is essential for the biosynthesis of the representative meroterpenoid furaquinocin M. By leveraging a CellFree Protein Synthesis (CFPS) platform, we found that Fur5 catalyzes the transformation of 8-amino-flaviolin (8-AF) into diazo-flaviolin, which subsequently undergoes non-enzymatic deamination to yield the downstream intermediate flaviolin. Our findings suggest that Fur5, together with the nitrite-generating enzymes Fur15 and Fur16, facilitates the deamination of 8-AF via diazotization in furaquinocin biosynthesis. We further identified the nitrite-binding pocket within Fur5 and proposed a catalytic mechanism in which nitrite is activated through adenylation. The findings unveil a diazo-forming enzyme that facilitates deamination in meroterpenoid biosynthesis.
Keywords: Meroterpenoid, biosynthesis, diazonium, enzyme, deamination.
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