The Study Of Biosynthetic Pathway And Physiological Function Of The Diisonitrile Natural Product SF2768

Isonitrile is a special chemical group that presents nucleophilic and electrophilic properties at the same time,and natural products possessing isonitrile groups usually exhibit bioactivities such antimicrobial,antimalaria,and cytotoxicity.From 1950 s to 2016,only about 200 isonitrile natural products were discovered and elucidated,the diisonitriles are more rare than that.Up to the February 2020,there are less than 10 groups of diisonitriles skeleton were reported.The research on their biosynthesis and mode of action are meaningful to bringing insights into microorganism interaction and drug design due to the bioactivities.Previously,a biosynthetic gene cluster of a diisonitrile natural product SF2768 was cloned from Streptomyces thioluteus DSM40027 by our group,the pathway was proposed by molecular biological and bioinformatics analysis.Meanwhile,SF2768 proved to be a chalkophore that can specifically bind and import environmental copper.In this study,we investigated the biosynthetic enzymes in the gene cluster of SF2768.The biosynthesis of the skeleton of SF2768 was verified by in vitro reconstitution of the four biosynthetic enzyme,Sfa B,Sfa C,Sfa D,and Sfa E and we found that the AMP-ligase Sfa B in the pathway was a promiscuous enzyme that can recognize different small molecule carboxylic acids.Taking advantage of this feature,we biosynthesized 30 unnatural SF2768 analogs for bioactivity screening.Furthermore,we found a rare isonitrile hydratase Sfa F in the biosynthetic pathway of SF2768,and the in vivo and in vitro results revealed that Sfa F can sequentially hydrate the two isonitrile groups of SF2768 and its diisonitrile analogs,thus eliminating their copper-chelating ability.We proposed that Sfa F is a self-resistance enzyme which can inactivate over-accumulated SF2768 in the cell of S.thioluteus to avoid copper metabolism disorder.The other proposed function is chemical defense for the isonitrile compound secreted by other microorganisms in environment.Homologous proteins of isonitrile hydratase Sfa F distribute widely in a variety of microorganisms,but few of them are investigated.This study for the first time proved the physiological function of isonitrile hydratase,thereby promoting the study of homologous hydratase in other microorganisms.Finally,we explored the antibacterial mechanism of SF2768.Through the results of a series of experiments including ICP-MS determination of the intracellular copper ion content,the intracellular ATP content,the level of the reactive oxygen species accumulation,and activity measurement of SF2768 analogs,we found that after incubation with SF2768,the cellular copper content of Bacillus subtilis was decreased.Meanwhile,the ATP synthesis level was decreased and the reactive oxygen species was increased significantly.We thus hypothesized that SF2768 binds catalytic copper of cytochrome c oxidase in the respiratory chain,which led to a large amount of reactive oxygen species that inhibits the growth of B.subtilis.In addition,SF2768 and its diisonitrile analogues were proved to be effective inhibitors of tyrosinase in vitro,which further confirmed that SF2768 is a copper-dependent enzyme inhibitor,supporting the hypothesis proposed in this study.Overall,we proved that the diisonitrile natural product SF2768 can not only assist in obtaining copper from the environment for streptomyces,but also produce oxidative stress on other microorganisms,thus bringing new insights of the function of isonitrile natural products.Through a series of assays,this study provides a case of microorganisms using special chemical functional groups to control transition metals,which offers a new understanding of the relationship between microorganisms,natural products and transition metals.