Preliminary Exploration On The Biosynthetic Mecahnism Of The Fluorene Ring And Diazo Group In Kinamycin

Kinamycin is a class of angucyclinone produced by type II polyketide synthase system.Structurally,it has three distinctive features:benzofluorene core,highly oxidized A ring,and a diazo group on fluorene ring.It is reported that all three functional groups are related to their biological activity.Therefore,many studies on the biosynthesis of kinamycin have centered on the formation of the above three functional groups.Due to its unique structure and good bioactivity,kinamycin has attracted a wide range of research interests.The key to the formation of benzofluorene structure is the formation of the fluorene ring.Previous studies have shown that in the biosynthetic pathway of kinamycin,Alp J and Alp K act synergistically to catalyze the opening and closure of the original aromatic ring,thereby forming a fluorene ring structure.In the mechanism presumed by the previous study,Alp J opened and closed the aromatic ring using FADH₂as cofactor provided by Alp K.In addition,Alp K hydroxylate benzofluorene intermediate,preventing the intermediate from aggregating and thus flowing out of the kinamycin biosynthetic metabolic pathway.In this study,the in vitro reconstituted enzyme-catalyzed reaction system was used to study the unique rearrangement involved in the kinamycin biosynthesis.The results provided some new insights into the reaction mechanism.Alp J could catalyze both the ring-opening and ring-closure reactions.In the absence of Alp K,Alp J may complete the hydroxylation of the benzofluorene intermediate.For the first time it was found that Alp J catalyze the substrate to produce two products without a cofactor.Through the structural determination of one of the products,it was further proved that Alp J does not require cofactors to open the aromatic ring.Because the other product was extremely unstable,the determination of its structure could not be completed,but many experimental clues indicated that Alp J can complete the subsequent ring-closure reaction without the need of a cofactor.So far,the biosynthetic pathway of kinamycin has not been completely elucidated,especially for the formation of the diazo group.Previous studies have been hampered by lack of information regarding complete biosynthetic gene cluster of kinamycin.Our group has recently identified the complete biosynthetic gene cluster of kinamycin,which contains 62 genes.Among them,eight genes have not been reported.Through bioinformatics analysis,we found that the alp2F and alp2G is highly homologous to cre E and cre D in cremeomycin biosynthetic pathway.The gene products encoded by cre E and cre D catalyze the production of nitrous acid from aspartic acid.The nitrous acid is spontaneously loaded onto the carbon skeleton to form the diazo group under acidic conditions.In this study,we confirmed that Alp2F and Alp2G can also catalyze the formation of nitrous acid from aspartic acid by in vitro enzyme-catalyzed reaction.Furthermore,sodium nitrite feeding experiment using alp2F and alp2G double mutant confirmed the involvement of nitrite in the kinamycin biosynthesis.The corroboration of the function of alp2F and alp2G both in vitro and in vivo is not only a characterization of the unknown genes in kinamycin biosynthetic gene cluster,but also paved the way for the elucidation of the formation of the diazo group.