Biosynthetic Mechanism Of The Biosynthetic Precursors Of Streptonigrin

Streptonigrin is a highly functionalized aminoquinone alkaloid produced by Streptomyces flocculus ATCC 13257.It shows in vivo and in vitro antiviral properties,potent and broad spectrum activities against bacteria and fungi as well as potent activity against a wide range of tumors.The antitumor mechanism studies have identified that streptonigrin induces DNA strand breaks and blocks the synthesis of DNA and RNA.The streptonigrin gene cluster was identified by genome scanning and a series of gene replacement stuies.The whole cluster contains 48 genes with stnA as its upstream boundary and stnT4 as the downstream boundary.Based on bioinformatics analysis,chemical structures of the obtained intermediates and feeding experiments,the biosynthetic pathway of streptonigrin has been proposed.This study is mainly focused on the enzymatic mechanism of the biosynthetic pathways of the two key biosynthetic precursors of streptonigrin: the ?-carboline moiety formed by condensation of(2S,3S)-?-methyltryptophan and erythrose-4-phosphate,and the multi-substituted benzoquinone moiety.(2S,3R)-?-methyltryptophan and its isomer(2S,3S)-?-methyltryptophan were biosynthesized by different combination of StnR(an aminotransferase),StnK3(a cupin protein),and StnQ1(a methykltransferase).StnR and StnQ1 catalyzed the formation of(2S,3R)-?-methyltryptophan,while StnR,StnK3,and StnQ1 together catalyzed the(2S,3S)-?-methyltryptophan formation.Most importantly,(2S,3S)-?-methyltryptophan was established as the real biosynthetic intermediate of the streptonigrin pathway by feeding ?stnQ1 mutant with these isomers,but not(2S,3R)-?-methyltryptophan that was previously stated.StnK2 has been characterized to be a new Pictet-Spenglerase in vitro.The biochemical characherization established that StnK2 stereoselectively catalyzes the Pictet-Spengler(PS)reaction of(2S,3S)-?-methyltryptophan and erythrose-4-phosphate to produce(1S)-tetrahydro-?-carboline scaffold,without any reducing co-factor.StnK2 exhibits a relatively broad substrate tolerance for L-tryptophan counterpart and can accept L-tryptophan derivatives with indole ring substituded,such as fluoro-substituted L-tryptophan as the substrates,but it shows strict substrate specificity for the erythrose-4-phosphate part.On the basis of bioinformatics,StnP2 is likely to be a flavin adenine dinucleotide(FAD)-dependent D-amino acid oxidase.Here,it was biochemically characterized to catalyze two-electron dehydrogenation of tetrohydro-?-carboline to generate dihydro-?-carboline,so it is named to be a ?-carboline synthase.StnP2 is a FAD dependent oxidase,catalyzes the oxidation of tetrohydro-?-carboline.It not only exhibits high stereoslectivety and only accepts(1S,3S)-tetrahydro-?-carboline as the substrate,but also flexible substrate specificity and can accept(1S,3S)-tetrahydro-?-carboline with different substituents at C1-position.Due to the different substituents at C1-position,the resulting dihydro-?-carbolines are spontaneously oxidized to ?-carboline accompanied with oxidation,dehydration and other reactions of the substituents at C1-position.This is the first time to reveal the enzymatic mechanism of aromatic degrees of ?-carboline existed in nature.The multi-substituted benzoquinone moiety is another important biosynthetic precursor of streptonigrin and is derived from 3-hydroxy anthranilic acid.Based on the comparation of the biosynthetic enzymes for streptonigrin and calcimycin which have the same scaffold,we proposed that six enzymes(StnI,J,H1,K1,L,and S1)may be involved in the C7 amination of streptonigrin.In vitro biochemical characterization confirms that StnK1 and StnL catalyze the formation of nitrious acid from L-aspartic,and isotope feeding experiments confirms that the C7 amino is derived from nitrous acid.These studies set the stage for the complete elucidation of the formation mechanism of C7 animo group of streptonigrin.The biosynthetic pathway of the two major biosynthetic precursors of streptonigrin may involve some novel enzymatic reactions.Elucidation of their enzymatic mechanisms not only paves a way for the complete understanding of streptonigrin biosynthesis,but also enrichs the diversity of biochemistry in nature.