Functional Characterization of a Diazo‐Forming Enzyme in Meroterpenoid Biosynthesis

A diazo-forming enzyme (stFur5) facilitates deamination in the biosynthesis of furaquinocins and other polyketide-derived meroterpenoids.

Meroterpenoids are known for their distinct structure and hybrid biosynthetic origin. The biosynthetic gene clusters 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, it is shown that one of these genes, stfur5, is essential for the biosynthesis of the representative meroterpenoid furaquinocin M. By leveraging a cell-free protein synthesis platform, it is found that stFur5 catalyzes the transformation of 8-amino-flaviolin (8-AF) into diazo-flaviolin, which subsequently undergoes nonenzymatic deamination to yield the downstream intermediate flaviolin. The findings suggest that stFur5, together with the nitrite-generating enzymes stFur15 and stFur16, facilitates the deamination of 8-AF via diazotization in furaquinocin biosynthesis. We further identified the nitrite-binding pocket within stFur5 and proposed a catalytic mechanism in which nitrite is activated through adenylation. The findings enrich the understanding of the role of diazo-forming enzymes in natural product biosynthesis.

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