Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-C...Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-Cy FE catalyzes the crosslinking between an aromatic residue(Ω1) and a non-aromatic residue(X3) in a Ω1-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the Ri PP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type triceptides, showing these Ri PPs are likely class-specific and only present in gamma-proteobacteria. The 3-Cy FE Pau B from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of Pau B in vitro,showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step.展开更多
Dynobactin A is a ribosomally synthesized and posttranslationally modified peptide(RiPP)antibiotic that displays potent activity against Gram-negative bacteria.This compound features a bicyclic scaffold consisting of ...Dynobactin A is a ribosomally synthesized and posttranslationally modified peptide(RiPP)antibiotic that displays potent activity against Gram-negative bacteria.This compound features a bicyclic scaffold consisting of a C–C crosslink between Trp1 and Asn4,and a highly unique N–C crosslink between His6 and Tyr8.In this study,we successfully reconstituted the activity of the radical S-adenosylmethionine(SAM)enzyme DynA both in vivo and in vitro,demonstrating that it catalyzes the formation of both C–C and N–C crosslinks in dynobactin A.Biochemical studies and mutagenesis analysis support that the N–C crosslink is formed via a para-quinone methide intermediate,representing an unprecedented mechanism in RiPP biosynthesis.We also showed DynA catalysis proceeds in a stepwise manner,where the N–C crosslink is formed first,followed by a C–C crosslink.These findings provide new insights into radical SAM enzyme chemistry and open avenues for engineering novel RiPP antibiotics targeting Gram-negative bacteria.展开更多
基金supported by grants from the National Key Research and Development Program (Nos. 2018YFA0900402 and 2021YFA0910501)the National Natural Science Foundation of China (Nos. 21822703, 21921003, and 32070050)+2 种基金the funding of Innovative research team of high-level local universities in Shanghaia key laboratory program of the Education Commission of Shanghai Municipality (No. ZDSYS14005)West Light Foundation of the Chinese Academy of Sciences (No. xbzgzdsys-202105)。
文摘Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-Cy FE catalyzes the crosslinking between an aromatic residue(Ω1) and a non-aromatic residue(X3) in a Ω1-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the Ri PP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type triceptides, showing these Ri PPs are likely class-specific and only present in gamma-proteobacteria. The 3-Cy FE Pau B from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of Pau B in vitro,showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step.
基金supported by grants from the National Natural Science Foundation of China(grant nos.22477049,21921003,32270070,32070050,and U22A20451)from West Light Foundation of the Chinese Academy of Sciences(grant no.xbzg-zdsys-202105).
文摘Dynobactin A is a ribosomally synthesized and posttranslationally modified peptide(RiPP)antibiotic that displays potent activity against Gram-negative bacteria.This compound features a bicyclic scaffold consisting of a C–C crosslink between Trp1 and Asn4,and a highly unique N–C crosslink between His6 and Tyr8.In this study,we successfully reconstituted the activity of the radical S-adenosylmethionine(SAM)enzyme DynA both in vivo and in vitro,demonstrating that it catalyzes the formation of both C–C and N–C crosslinks in dynobactin A.Biochemical studies and mutagenesis analysis support that the N–C crosslink is formed via a para-quinone methide intermediate,representing an unprecedented mechanism in RiPP biosynthesis.We also showed DynA catalysis proceeds in a stepwise manner,where the N–C crosslink is formed first,followed by a C–C crosslink.These findings provide new insights into radical SAM enzyme chemistry and open avenues for engineering novel RiPP antibiotics targeting Gram-negative bacteria.
