Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide(RiPP)superfamily.In this study,a genome mining approach led to the ident...Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide(RiPP)superfamily.In this study,a genome mining approach led to the identification of a novel linaridin,mononaridin(MON),from Streptomyces monomycini.In-frame deletion genetic knockout studies showed that,in addition to many genes essential for MON biosynthesis,monM encodes an S-adenosyl methionine(SAM)-dependentα-N-methyltransferase that is responsible for installing two methyl groups in the MON N-terminus.Besides SAM,MonM also accepts ethyl-SAM and allyl-SAM,in which the methyl of SAM is replaced by an ethyl and an allyl,respectively.We showed that ethyl-SAM and allyl-SAM have distinct reactivities in MonM catalysis,and this observation was further investigated in detail by density functional theory(DFT)calculations.Remarkably,MonM acts efficiently on nisin,a prototypic lantibiotic that is structurally very different from the native substrate,and the ability of MonM to transfer an allyl group to the nisin N-terminus allowed production of a fluorescently labeled nisin,which can be further used in microscopic cell analysis.Our studies provide new insights into linaridin biosynthesis and demonstrate the potential of linaridin methyltransferases in bioengineering applications.展开更多
The linaridin antibiotic cypemycin is a ribosomal synthesized and post-translationally modified peptide(RiPP)that possesses potent activity against mouse leukemia cells.This peptide natural product contains an S-[(Z)-...The linaridin antibiotic cypemycin is a ribosomal synthesized and post-translationally modified peptide(RiPP)that possesses potent activity against mouse leukemia cells.This peptide natural product contains an S-[(Z)-2-aminovinyl]-D-cysteine(AviCys)moiety in the C-terminus.Formation of AviCys moiety requires an oxidative decarboxylation of the C-terminal Cys of the precursor peptide CypA,and this process is catalyzed by a flavin-containing protein CypD.In this work,we tested CypD substrate specificity with a series of synthetic oligopeptides.We show that most of the N-terminal sequence of CypA is not required for CypD activity,and the Cterminal three residues serve as the minimal structural element for enzyme recognition.We also show that CypD tolerates various substrates with modified C-termini,allowing for the generation of four novel cypemycin variants with modified AviCys moiety by site direct mutagenesis of the precursor peptide CypA.Our study demonstrates the relaxed substrate specificity of CypD and lays a foundation for future bioengineering of AviCys-containing natural products.展开更多
基金supported in part by grants from National Natural Science Foundation of China(21822703 and 31670060 to Q.Z.,21803030 to Y.L.,and 31600398 to W.D.)from the National Key Research and Development Program(2018Y F A0900402 and 2016 Y F A0501302)from Shanghai Key Laboratory of Bioactive Small Molecules(ZDSYS14005)。
文摘Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide(RiPP)superfamily.In this study,a genome mining approach led to the identification of a novel linaridin,mononaridin(MON),from Streptomyces monomycini.In-frame deletion genetic knockout studies showed that,in addition to many genes essential for MON biosynthesis,monM encodes an S-adenosyl methionine(SAM)-dependentα-N-methyltransferase that is responsible for installing two methyl groups in the MON N-terminus.Besides SAM,MonM also accepts ethyl-SAM and allyl-SAM,in which the methyl of SAM is replaced by an ethyl and an allyl,respectively.We showed that ethyl-SAM and allyl-SAM have distinct reactivities in MonM catalysis,and this observation was further investigated in detail by density functional theory(DFT)calculations.Remarkably,MonM acts efficiently on nisin,a prototypic lantibiotic that is structurally very different from the native substrate,and the ability of MonM to transfer an allyl group to the nisin N-terminus allowed production of a fluorescently labeled nisin,which can be further used in microscopic cell analysis.Our studies provide new insights into linaridin biosynthesis and demonstrate the potential of linaridin methyltransferases in bioengineering applications.
基金the National Key Research and Development Program(2016 YF A0501302 to Q.Z.)from National Natural Science Foundation of China(31600398 to W.D.,and 31500028 and 31670060to Q.Z.)State Key Laboratory of Microbial Technology Open Projects Fund(M2015-01 to W.D.).
文摘The linaridin antibiotic cypemycin is a ribosomal synthesized and post-translationally modified peptide(RiPP)that possesses potent activity against mouse leukemia cells.This peptide natural product contains an S-[(Z)-2-aminovinyl]-D-cysteine(AviCys)moiety in the C-terminus.Formation of AviCys moiety requires an oxidative decarboxylation of the C-terminal Cys of the precursor peptide CypA,and this process is catalyzed by a flavin-containing protein CypD.In this work,we tested CypD substrate specificity with a series of synthetic oligopeptides.We show that most of the N-terminal sequence of CypA is not required for CypD activity,and the Cterminal three residues serve as the minimal structural element for enzyme recognition.We also show that CypD tolerates various substrates with modified C-termini,allowing for the generation of four novel cypemycin variants with modified AviCys moiety by site direct mutagenesis of the precursor peptide CypA.Our study demonstrates the relaxed substrate specificity of CypD and lays a foundation for future bioengineering of AviCys-containing natural products.
