Rishirilide A and B, first isolated in 1984 during a α2-macroglobulin inhibitor screen, belong to the class of tricyclic aromatic polyketides and are produced by multiple Streptomyces strains (1). Labeling studies validated the polyketide synthase-dependent biosynthesis and already suggested an unusual backbone rearrangement of the isopentyl substituent (2). Knockout studies in the rishirilide producer Streptomyces bottropensis in combination with in vitro enzyme reactions revealed RslO9, a FAD-dependent Monooxygenase (FPMO), as the key player in the last steps of rishirilide biosynthesis (3). However, the catalytic mechanism of this biosynthesis leading to rishirilide A/B and lupinacidin A remains unknown. Unraveling the intriguing activity could give insights into the structural diversification of aromatic polyketides and FPMO chemistry.

For in depth analysis, RslO9 and N-terminal deletion variants were cloned and heterologously expressed, followed by purification and structure elucidation by X-ray crystallography. The molecular structure of RslO9 was obtained at a resolution of 2.4 Å. Cocrystallization attempts with the native substrate prevented formation of crystals, while soaking experiments revealed a conformational change in the active site, although no clear electron density for the substrate could be observed. Activity assays with numerous substrate-like compounds showed conversion of multiple naphthoquinones and implied a facilitating effect of alkyl substituents. On the other hand, anthraquinone derivatives were not converted, suggesting a terminal paraquinone moiety as an important feature for catalysis. Furthermore, docking and molecular dynamics simulations as well as site-directed mutagenesis studies are ongoing to pin down essential residues for substrate binding and catalysis.

In conclusion our studies give insights into the backbone rearrangement performed by RslO9, further deepening our understanding of flavoprotein chemistry and polyketide tailoring reactions.

References

[1] Hideyuki Iwaki et. al. Structures of rishirilides A and B, alpha 2-macroglobulin inhibitors produced by Streptomyces rishiriensis OFR-1056. J. of Antibiotics 1984; 34(9):1091–3.

[2] Philipp Schwarzer et. al. Biosynthesis of rishirilide b. Antibiotics. 7. März 2018;7(1).

[3] Olga Tsypik et al. Oxidative Carbon Backbone Rearrangement in Rishirilide Biosynthesis. J Am Chem Soc. 2020;142(13):5913–7.