Rubromycins are a class of aromatic polyketides with potent antibiotic properties and are produced by a wide variety of actinobacteria. A distinctive feature of rubromycins is the presence of a [5,6]‑spiroketal moiety at the core of the molecule. Previous work in our group elucidated the mechanism behind the initial formation of a [6,6]‑spiroketal unit during rubromycin maturation through the concerted action of GrhO1 and GrhO5.¹ The resulting intermediate is subsequently transformed by the flavoprotein monooxygenase GrhO6, leading to the contraction of ring C to form the [5,6]‑spiroketal structure found in mature rubromycins. Interestingly, efficient ring contraction only takes place in presence of GrhJ, which belongs to the family of GCN5‑related N‑acetyltransferases (GNAT).^2,3 However, the precise catalytic mechanism of GrhO6 and the role of GrhJ in this process remain to be elucidated. Here, we aimed to investigate the catalytic mechanism of GrhO6 and the role of GrhJ in the [5,6]‑spiroketal formation by recombinantly producing these proteins and employing a combination of biochemical assays, biophysical methods, and X‑ray crystallography. The crystal structure of GrhO6, together with biochemical and mutagenesis studies, indicates that a potential acetylation of GrhO6 by GrhJ is unlikely to be responsible for the spiroketal ring contraction. Furthermore, differential scanning fluorimetry data indicate that GrhJ binds to rubromycin-type molecules, despite the absence of N‑acetylation sites in rubromycins or their biosynthetic intermediates. We propose that GrhJ may not act as an acetyltransferase. Instead, it might facilitate the release of the product from GrhO6 or stabilize reaction intermediates to minimize side reactions during spiroketal ring contraction. These insights advance our understanding of rubromycin biosynthesis and suggest new functional roles for members of the GNAT family. Further investigation is required to elucidate the detailed mechanism by which GrhJ promotes this reaction.

References

[1] Toplak et al. Angew Chem Int Ed. 2021 Dec 20;60(52):26960–70.

[2] Yunt et al. J Am Chem Soc. 2009 Feb 18;131(6):2297–305.

[3] Toplak et al. Chem Sci. 2022;13(24):7157–64.