Mechanism Of The Carbon Skeleton Rearrangement In The Biosynthesis Of Phenanthraquinone Antibiotic Murayaquinone

Carbon skeleton rearrangement is crucial for the structural diversity and biological activity of natural products,thus great attention has been devoted to identifying the relevant enzymes and cleavage mechanisms.Owning to the unstablility of the intermediates in the rearrangement processes,seldom mechanism has been unambiguously illucidated.Angucyclines from bacteria are synthesized by type ? PKS either through cyclized directely from poly-?-keto chain or from carbon skeleton rearrangement.However,little biochemical evidence has been obtained to support this kind of rearrangement.Murayaquinone,with the angular skeleton of 9,10-phenanthraquinone,is synthesized by bacterial type ? PKS and experiences skeleton rearrangement confirmed by early isotope experiment.In this study,the gene cluster which is silent in the original strain was identified employing a heterologous expression system.The rearrangement mechanism was studied.The boundaries of the gene cluster and genes essential for murayaquinone assembly were identified.The gene cluster includes a biosynthesis region and a regulation region which is 28 kb apart.The biosynthesis region contained 3 minimal PKS genes(mrqA,mrqB,mrqC),3 ketoreductase genes(mrqM,mrqF,mrqH),3 cyclase genes(mrqD,mrqE,mrqJ),8 oxidase genes(mrqO1-O8)and 4 hyperthetical genes(mrqG,mrqK,mrqL,mrqI).In order to elucidate the rearrangement mechanism,each single biosynthesis gene was knocked out.Three anthraquinone-type of linear aromatic compounds,including an anthracenone epoxide,were identified from some of the mutants.In order to determine whether they were intermediates or shunt products,these compounds were fed to ?PKS mutant.Murayaquinone was produced when the linear anthracenone epoxide was fed to ?PKS thus it was indicated to be an intermediate of the biosynthetic pathway.The accumulation of the anthracenone epoxide in ?mrqO7(a lot)and ?mrqO6(trace amounts)suggested that MrqO7 and MrqO6 played pivotal roles in the formation of murayaquinone from the anthracenone epoxide intermediate.To further determine whether other genes were also involved in the conversation of the anthracenone epoxide into murayaquinone,the anthracenone epoxide was fed to mutants that abolished the production of murayaquinone and did not produce this anthracenone epoxide.Murayaquinone was detected when the anthracenone epoxide was fed to all these mutants except for ?mrqO3,revealing that MrqO3,maybe along with MrqO6 and MrqO7,is responsible for the conversion of this anthracenone epoxide into murayaquinone.Sequence analysis revealed that MrqO7 was a putative NAD(P)H-dependent oxidoreductase,MrqO6 was an FAD-dependent monooxygenase and MrqO3 was a homologue of cholesterol oxidase.To uncover the mechanism of MrqO3,MrqO6 and MrqO7 that catalyze the conversion of the anthracenone epoxide into murayaquinone,an in vitro assay system containing MrqO3,MrqO6,and MrqO7 in the presence of NADPH or NADH using the anthracenone epoxide as a substrate was designed.The three enzymes could completely convert the anthracenone epoxide to murayaquinone while any one of the three enzymes was removed from the assay,murayaquinone was not detected anymore.The sequence of events involving each enzyme was investigated,using each enzyme individually in the presence of NADPH and the anthracenone epoxide.The results indicated that both MrqO6 and MrqO7 could recognize the anthracenone epoxide as substrate.The reaction with MrqO3 did not show any conversion of the substrate,indicating that MrqO3 catalyzed the following last step of the conversion of the anthracenone epoxide to murayaquinone.According to LC-MS analysis,the product catalyzed by MrqO6 was 16 Da more than that of the anthracenone epoxide,indicating that one oxygen atom was inserted into the anthracenone epoxide while the product catalyzed by MrqO7 was 2 Da more than that of the anthracenone epoxide,indicating an addition of two hydrogen atoms.To further clarify the catalytic order involving MrqO7 and MrqO6 during the conversion of the anthracenone epoxide to murayaquinone,three parallel sequential reactions were carried out.It was clearly demonstrated that the reductive epoxide opening of the anthracenone epoxide catalyzed by MrqO7 occurs prior to the MrqO6-catalyzed Baeyer-Villiger reaction.The rearrangement process was proposed as follows: MrqO7-catalyzed hydrogenation on the anthracenone epoxide which opened epoxide is the first step,followed by Baeyer-Villiger oxidation,carbon bond cleavage and new carbon bond reformation catalyzed by MrqO6.Finally MrqO3 may catalyze oxidative and reductive reactions,accompanying the loss of 2 molecules of water,eventually to form the final product murayaquinone.To date,this anthracenone epoxide was the first verified intermediate in the rearrangement of linear aromatic polyketide to angular aromatic polyketide.And this kind of rearrangement was first proved by the in vitro enzymatic assay that MrqO7,MrqO6 and MrqO3 cooperatively catalyze the conversion of an anthracenone epoxide to murayaquinone.Overall,the studies revealed a novel strategy for the biosynthesis of angular aromatic polyketides in nature.