Pure merchandise containing xanthone scaffold confirmed numerous pharmacological properties, comparable to anti-inflammatory, anti-oxidant, anti-microbial, and anti-tumor actions. Quite a few xanthones have been remoted from completely different sources for the reason that first xanthone compound reported 4 decays in the past. Various strategies have additionally been developed for the chemical synthesis of xanthones. Nevertheless, the biosynthetic mechanisms of the xanthone formation haven’t been nicely documented.
Fig. 1 The physiological and sudden capabilities of the multi-functional monooxygenase FlsO1.
Fluostatin C (FST C, 7) is a marine-actinomycete (Micromonospora rosaria SCSIO N160) derived compound and belongs to benzofluorene-containing atypical angucyclines, the identical class of kinamycin A. Throughout our biosynthetic research on FSTs, the monooxygenase FlsO2 was biochemically characterised by us to effectively convert prejadomycin (PJM, 8) to dehydrorabelomycin (DHR, 9) by CR1 (10) (Fig. 1). To our shock, the manufacturing of DHR (9) was nonetheless noticed within the flsO2-inactivation mutant of M. rosaria SCSIO N160, though in minor quantity (Org. Lett. 17, 5324, 2015). Given the presence of a number of extremely related monooxygenases within the FST biosynthetic gene cluster, we then hypothesized that one among these monooxygenases may compensate for the operate of FlsO2 to transform 8 to 9. Within the meantime, Professor Keqian Yang’s group, Institute of Microbiology, Chinese language Academy of Sciences, reported that the monooxygenase AlpJ (a homologue of FlsG) catalyzed an oxidative B-ring cleavage and contraction to transform DHR (9) putatively to benzo[b]-fluorene intermediate (12) (Fig. 1). Additionally they proposed that the flavoenzyme AlpK in all probability additional hydroxylated 12 at C-5 to hydroquinone-kinobscurinone (13) (Chem. Commun. 51, 8845, 2015), which was not directly confirmed by the Balskus’s group in a coupling response of AlpJK (J. Am. Chem. Soc. 139, 2864, 2017). Final 12 months, we additionally carried out the in vivo characterization of the flavoprotein monooxygenase (FPMO) FlsO1 because the C-5 hydroxylase that putatively transformed 12 to 13 (Fig. 1), functionally equal to its homologous enzyme AlpK (J. Org. Chem. 86, 11019, 2021). Nevertheless, the direct in vitro biochemical characterization of FlsO1 and AlpK was hampered by the inavailability of the extremely instable substrate 12. Apparently, we just lately remoted an intermediate nenestatin C (14) (Fig. 1) from the biosynthetic pathway of nenenestatin A (Org. Biomol. Chem. 19, 4243, 2021), which had the identical carbon scaffold as 12 and will function a possible substrate mimic of 12.
To handle whether or not one other FPMO within the FST pathway can complement the operate of FlsO2, we overproduced 4 enzymes FlsO1, FlsO3, FlsO4 and FlsO5 to probe their exercise towards 8. Though FlsO3‒O5 are inactive, to our delight, FlsO1 certainly can effectively convert 8 to 9. This remark satisfies our curiosity to search out the enzyme sharing the identical operate as FlsO2. Extra impressively, FlsO1 not solely catalyzes the conversion of 8 to 9, but in addition is able to reworking 8 into a number of merchandise, that are characterised as xanthone-containing merchandise 16 and 17 and the C-ring opening compound 18 (Fig. 1). It is a bona fide sudden discovering. We then carried out a collection of experiments, together with time course examine on the FlsO1 response with 8, intermediates/shunt merchandise characterization, and isotope-labeling research (18O2 and MeOH-d4) to finally led to an-experiment-data supported speculation of the FlsO1-catalyzed xanthone formation. It includes three-step successive oxidations (together with hydroxylation, epoxidation and Baeyer-Villiger oxidation) (Fig. 2). In short, FlsO1 first catalyzes C-12 hydroxylation of PJM (8) to 19, which spontaneously turns into 20, which might bear spontaneous dehydration to 9. Then, FlsO1 catalyzes a second oxidation, specifically epoxidation of 20, to afford 22. Subsequently, FlsO1 catalyzes a 3rd oxidation, a Baeyer-Villiger response of 22, resulting in 29. Lastly, 29 can bear a collection of spontaneous reactions to finally give rise to the xanthone merchandise 16 by way of two putative routes (routes 1 and 2, Fig. 2).
Subsequent, we use nenestatin C (14), mimicking the pure substrate 12, to probe the physiological operate of FlsO1. Certainly, FlsO1, and its homologous enzymes AlpK from the kinamycin pathway and Nes26 from the nenestatin pathway, had been confirmed to effectively catalyze the C-5 hydroxylation of 14, offering direct in vitro proof to help their in vivo operate. Final however not the least, the substrate flexibility and multifunction of FlsO1 could be defined by a broad substrate binding cavity by resolving the crystal construction of FlsO1 in advanced with the cofactor flavin adenine dinucleotide (FAD). In short, this examine assist growing the sights of versatile capabilities of flavoproteins.
Fig. 2 The enzymatic mechanism of FlsO1-catalyzed xanthone formation.
Ultimately, I additionally need to share a few of my private experiences within the current work. This work is a part of our long-term pursuit purpose to know the biosynthesis of atypical angucyclines and to develop their structural variety thereby. As an Early Profession Researcher, I’ve learnt rather a lot since being concerned on this venture. As soon as the analysis venture has been in fingers, extra attentions and endurance ought to be paid, similar to caring for a brand new child. Undoubtly, one will meet many troubles, obstacles and even failures in the way in which of attaining the analysis purpose. It’s essential to maintain calm, considerate and chronic. One ought to be very cautious in analyzing particulars of every experiment and will take rapid actions to resolve the issues. Furthermore, one ought to be curious for any irregular experimental phenomenon. Any sudden discovery shouldn’t be ignored and we must always strive our greatest to probe for extra hidden info behind the irregular discovery. Actually, it’s the bizarre presence of compound 9 within the ∆flsO2 mutant resulting in the invention of sudden operate of FlsO1. Generally, we really feel annoyed by the unstable response intermediates, the unclear NMR alerts or the undesired HPLC profiles. Nevertheless, we by no means cease and quit what we do.