WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 ...WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.展开更多
基金supported by the BBSRC(MIBTP studentship to Daniel J.Leng)the Monash Warwick Alliance(Seed Fund Award to Manuela Tosin and Max J.Cryle)+6 种基金the University of Warwick(Career Support Award to Manuela Tosin)Monash University,EMBL Australia,the Australian Research Council(Discovery Project DP210101752 to Max J.Cryle)the National Health and Medical Research Council(APP1140619 to Max J.Cryle)the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science(CE200100012)funded by the Australian Governmentfunded by the National Natural Science Foundation of China(82104044 to Songya Zhang)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-PTJS-003-07)。
文摘WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.
