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The B1 Protein Guides the Biosynthesis of a Lasso Peptide.

ABSTRACT: Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold that endows them with extraordinary stability and biologically relevant activity. However, the biosynthetic mechanism of these fascinating molecules remains largely speculative. Generally, two enzymes (B for processing and C for cyclization) are required to assemble the unusual knot-like structure. Several subsets of lasso peptide gene clusters feature a "split" B protein on separate open reading frames (B1 and B2), suggesting distinct functions for the B protein in lasso peptide biosynthesis. Herein, we provide new insights into the role of the RiPP recognition element (RRE) PadeB1, characterizing its capacity to bind the paeninodin leader peptide and deliver its peptide substrate to PadeB2 for processing.

SUBMITTER: Zhu S

PROVIDER: S-EPMC5067487 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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The B1 Protein Guides the Biosynthesis of a Lasso Peptide.

Zhu Shaozhou S Fage Christopher D CD Hegemann Julian D JD Mielcarek Andreas A Yan Dushan D Linne Uwe U Marahiel Mohamed A MA

Scientific reports 20161018

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold that endows them with extraordinary stability and biologically relevant activity. However, the biosynthetic mechanism of these fascinating molecules remains largely speculative. Generally, two enzymes (B for processing and C for cyclization) are required to assemble the unusual knot-like structure. Several subsets of lasso peptide gene clusters feature a "s ...[more]

PMID: 27752134

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Leader peptide removal in lasso peptide biosynthesis based on penultimate isoleucine residue.

Project description:Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.

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A prevalent peptide-binding domain guides ribosomal natural product biosynthesis.

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Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance.

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Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.

Project description:Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

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