The Demethoxylation-Dependent Enzymatic Formation Of Xanthone Ring In Xantholipin Biosynthesis

Xantholipin,a member of polycyclic xanthone antibiotics,possesses a highly oxygenated angular fused hexacyclic molecular skeleton and features the unique xanthone ring nucleus.Xantholipin shows potent antibacterial,antifungal and antitumor activities,and the biosynthetic mechanism of xanthone ring has attracted widespread attention in scientific community.The biosynthetic gene cluster has been cloned and a preliminary biosynthetic pathway has been proposed based on the genetics research of several key biosynthetic genes.However,the molecular mechanism of xanthone ring formation is still obscure.Moreover,the biosynthesis of xantholipin involves other post-assembly tailoring steps such as methylation,chlorination and oxygenation,which have not been elucidated up to now.Hence,this study aims to clarify the biosynthetic mechanism of xanthone ring and illustrate multiple post-assembly modifications during xantholipin biosynthesis.These studies will not only improve the understanding of xantholipin biosynthetic pathway but also set stage for the construction of more effective derivatives.The mechanism of xanthone ring formation was firstly demonstrated.The Baeyer-Villiger monooxygenase,XanO4,catalyzed the formation of xanthone ring in the anthraquinone intermediate 5,accumulated from ?xanO4 mutant.Meanwhile,data analysis of in vitro isotopic experiments with labeled oxygen showed that the xanthone ring formation reaction involves sequential insertion of two oxygen atoms,accomplishing oxidative replacement of the carbonyl group and the methoxyl group in compound 5,which suggesting an unexpected cryptic oxidative demethoxylation reaction.In addition,studies of Arx30,a homologous protein of XanO4,proved the generality of XanO4-mediated reaction among the biosynthesis of other polyc yclic xanthone antibiotics.Based on sequence alignment,the arginine(R45)of XanO4 and Arx30 was replaced by alanine.In vitro enzymatic assay proved the necessity of R45 for xanthone ring formation.Meanwhile,XanO4R45 A and Arx30R45 A catalyzed the transformation of 5 into a demethoxylaed anthraquinone analogue 5f,enzymatic assays of which illustrated the indispensability of the methoxyl group at C17 in 5 for the formation of xanthone ring.The methoxyl group at C17 is essential for the formation of the methylenedioxy bridge in xantholipin,whereas XanO4 catalyzed the demethoxylation at C17 in 5,resulting in a newly formed hydroxyl group at C17 in compound 12.All these suggested the existence of a cryptic remethlation of the hydroxyl group at C17 during xantholipin biosynthesis.Studies of the three methyltransferases in xantholipin biosynthetic gene cluster contributed to the unveiling of methylation tailoring modifications.XanM1 may catalyze the methylation of the hydroxyl group at C17 in a unknown precursor compound of 5,while XanM2 is responsible for the methylation of the hydroxyl group at C13 after the formation of xanthone ring and before the formation of methylenedioxy bridge and XanM3 catalyze the cryptic remethylation of the hydroxyl group at C17,just following the demethoxylation coupled with xanthone ring formation.Ultimately,the in vitro enzymatic assay of the sole halogenase XanH supported the indispensibility of xanthone ring and methoxyl group at C17 for other post-assembly modifications.In conclusion,we demonstrated the enzymatic xanthone ring formation catalyzed by XanO4 and unveiled a novel cryptic demethoxylation coupled mechanism for the transformation of cyclic ketone to cyclic ether,different from the typical BV oxidation of ketone into lactone or ester catalyzed by Baeyer-Villiger monooxygenases.Research of multifunctional oxygenase XanO4 expands the repertoire of Baeyer-Villiger oxidations,and sets a good example for the biological synthesis of more effective xanthone derivatives.Meanwhile,functional research of three methyltransferases and halogenase proved the cryptic remethylation of the hydroxyl group at C17 and illustrated the necessity of xanthone ring and methoxyl group for other post-assembly modifications during xantholipin biosynthesis.Our work established solid foundation for further illustration of xantholipin biosynthetic pathway and would make substantial contribution to rational modification aiming at developing more effective xantholipin derivatives.