scholarly journals Leader peptide exchange to produce hybrid, new-to-nature ribosomal natural products

2021 ◽  
Author(s):  
Laura Franz ◽  
Jesko Koehnke
Keyword(s):  
Natural Products ◽  
Leader Peptide ◽  
Sortase A ◽  
Peptide Modifications ◽  
Peptide Exchange ◽  
Proof Of Principle

Ribosomal natural products contain exquisite post-translational peptide modifications that are installed by a range of pathway-specific enzymes. We present proof of principle for a Sortase A-based approach that enables peptide...

scholarly journals Co-Occurrence of Enzyme Domains Guides the Discovery of an Oxazolone Synthetase

2020 ◽  
Author(s):  
Tristan de Rond ◽  
Julia E. Asay ◽  
Bradley S. Moore
Keyword(s):  
Natural Products ◽  
Gene Annotation ◽  
Functional Characterization ◽  
Genome Mining ◽  
Catalytic Activities ◽  
Single Protein ◽  
Multifunctional Enzymes ◽  
Synthetic Intermediates ◽  
Functional Gene Annotation ◽  
Proof Of Principle

AbstractMultidomain enzymes are cellular machines that orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Our understanding of these enzymes’ capabilities drives progress in fundamental metabolic research, biocatalysis, and human health. Here, we report the development of a new genome mining approach for the targeted discovery of novel biochemical transformations through the analysis of co-occurring enzyme domains (CO-ED) in a single protein. CO-ED was designed to identify unannotated multifunctional enzymes for functional characterization and discovery based on the premise that linked enzyme domains have evolved to function collaboratively. Guided by CO-ED, we targeted an unannotated predicted ThiF-nitroreductase di-domain enzyme found in more than 50 proteobacteria. Through heterologous expression and biochemical reconstitution, we discovered a series of new natural products containing the rare oxazolone (azlactone) heterocycle and characterized the di-domain enzyme as the first reported oxazolone synthetase in biology. This enzyme has the potential to become a valuable biocatalyst for the production of versatile oxazolone synthetic intermediates. This proof-of-principle experiment validates CO-ED-guided genome mining as a new method with potential broad utility for both the discovery of novel enzymatic transformations and the functional gene annotation of multidomain enzymes.TOC graphic

scholarly journals Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition

2017 ◽  
Vol 114 (39) ◽  
pp. 10420-10425 ◽  
Author(s):  
Katherine M. Davis ◽  
Kelsey R. Schramma ◽  
William A. Hansen ◽  
John P. Bacik ◽  
Sagar D. Khare ◽  
...  
Keyword(s):  
Natural Products ◽  
Conformational Changes ◽  
Posttranslational Modification ◽  
Biologically Active ◽  
Leader Peptide ◽  
Cross Link ◽  
X Ray Crystallography ◽  
Recognition Element ◽  
Terminal Domain ◽  
Precursor Peptide

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine–tryptophan cross-link, which is installed by the radical S-adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon–carbon cross-link in the streptide family of natural products.

Synthetic analogs of indole-containing natural products as inhibitors of sortase A and isocitrate lyase

2010 ◽  
Vol 20 (23) ◽  
pp. 6882-6885 ◽  
Author(s):  
Yeon-Ju Lee ◽  
Yu-Ri Han ◽  
Wanki Park ◽  
Seo-Hee Nam ◽  
Ki-Bong Oh ◽  
...  
Keyword(s):  
Natural Products ◽  
Isocitrate Lyase ◽  
Sortase A ◽  
Synthetic Analogs

scholarly journals Catalytic Use of a Leader Peptide in the Biosynthesis of 3-Thiaglutamate

2018 ◽  
Author(s):  
Michael A. Funk ◽  
Chi Ting ◽  
Wilfred A. van der Donk
Keyword(s):  
Natural Products ◽  
Pseudomonas Syringae ◽  
Small Molecule ◽  
Cysteine Residue ◽  
Leader Peptide ◽  
Genome Sequences ◽  
Small Peptide ◽  
Biochemical Processes ◽  
C Terminus ◽  
Ribosomal Protein Synthesis

AbstractSmall molecule natural products are key modulators of many types of intra- and interspecies communication. The availability of genome sequences allows the discovery of pathways to previously unknown natural products. We describe here a pathway in which a ribosomally synthesized small peptide serves as a catalytic scaffold on which a small-molecule anti-metabolite is biosynthesized in Pseudomonas syringae. First, a cysteine residue is transferred from Cys-tRNA to the C-terminus of the peptide, a reaction that replaces ribosomal protein synthesis. Then, a translocation of the cysteine thiol from the β-carbon to the α-carbon is catalyzed by an oxidase that removes the β-carbon as formate. The resulting thiol is carboxymethylated and proteolysis releases 3-thiaglutamate, in the process regenerating the peptide scaffold. This pathway features three previously unknown biochemical processes.

scholarly journals Current Advancements in Sactipeptide Natural Products

2021 ◽  
Vol 9 ◽  
Author(s):  
Yunliang Chen ◽  
Jinxiu Wang ◽  
Guoquan Li ◽  
Yunpeng Yang ◽  
Wei Ding
Keyword(s):  
Natural Products ◽  
Biological Activities ◽  
Cysteine Residue ◽  
Leader Peptide ◽  
Sequencing Technology ◽  
Chemical Structures ◽  
Precursor Peptide ◽  
Modified Peptides ◽  
Core Peptide ◽  
Alpha Carbon

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products that benefited from genome sequencing technology in the past two decades. RiPPs are widely distributed in nature and show diverse chemical structures and rich biological activities. Despite the various structural characteristic of RiPPs, they follow a common biosynthetic logic: a precursor peptide containing an N-terminal leader peptide and a C-terminal core peptide; in some cases,a follower peptide is after the core peptide. The precursor peptide undergoes a series of modification, transport, and cleavage steps to form a mature natural product with specific activities. Sactipeptides (Sulfur-to-alpha carbon thioether cross-linked peptides) belong to RiPPs that show various biological activities such as antibacterial, spermicidal and hemolytic properties. Their common hallmark is an intramolecular thioether bond that crosslinks the sulfur atom of a cysteine residue to the α-carbon of an acceptor amino acid, which is catalyzed by a rSAM enzyme. This review summarizes recent achievements concerning the discovery, distribution, structural elucidation, biosynthesis and application prospects of sactipeptides.

scholarly journals Directed Transfer of Large DNA Fragments betweenStreptomyces Species

2000 ◽  
Vol 66 (5) ◽  
pp. 2274-2277 ◽  
Author(s):  
Zhihao Hu ◽  
David A. Hopwood ◽  
Chaitan Khosla
Keyword(s):  
Natural Products ◽  
Gene Cluster ◽  
High Frequency ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Dna Fragments ◽  
Challenging Problem ◽  
Chromosomal Dna ◽  
Large Gene ◽  
Proof Of Principle

ABSTRACT The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire ∼22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.

Targeted discovery of bioactive natural products using a pharmacophoric deconvolution strategy: Proof of principle with eleganolone from Bifurcaria bifurcata R. Ross

2018 ◽  
Vol 26 ◽  
pp. 138-142 ◽  
Author(s):  
Flore Nardella ◽  
Laure Margueritte ◽  
Barbara Lamure ◽  
Justine Martine Pierrette Viéville ◽  
Mélanie Bourjot
Keyword(s):  
Natural Products ◽  
Bioactive Natural Products ◽  
Bifurcaria Bifurcata ◽  
Strategy Proof ◽  
Proof Of Principle

scholarly journals Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

2020 ◽  
Author(s):  
graham a hudson ◽  
Annie R. Hooper ◽  
Adam J. DiCaprio ◽  
David Sarlah ◽  
Douglas Mitchell
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Structure Prediction ◽  
Gene Clusters ◽  
Leader Peptide ◽  
Biosynthetic Gene Clusters ◽  
New Class ◽  
Precursor Peptide ◽  
Modified Peptides ◽  
Subsequent Elimination

<p>Owing to advances in genomic sequencing and bioinformatics, the breadth of natural product biosynthetic gene clusters (BGCs) has meteorically risen. This remains true for ribosomally synthesized and post-translationally modified peptides (RiPPs), where the rate of bioinformatically identifying clusters vastly outpaces characterization efforts. Uniting bioinformatics and enzymological knowledge to predict the chemical product(s) of a RiPP BGC with total chemical synthesis to obtain the natural compound is an effective platform for investigating cryptic gene clusters. Herein, we report the bioinformatic identification of a biosynthetically divergent class of RiPP bearing a subset of enzymes involved in thiopeptide biosynthesis. These natural products were predicted based on BGC architecture to undergo a formal, enzymatic [4+2]-cycloaddition with subsequent elimination of the leader peptide and water to produce a tri-substituted pyridine-based macrocycle. Bearing a pyridine similar to thiopeptides but lacking the ubiquitous thiazole heterocycles, these new RiPPs were termed pyritides. One of the predicted natural products was chemically synthesized using an 11-step synthesis. This structure was verified to be chemically identical by an orthogonal chemoenzymatic synthesis utilizing the precursor peptide and the cognate [4+2]-cycloaddition enzyme. The chemoenzymatic platform was used to synthesize a second in-cluster pyritide product as well as analogs from other bioinformatically identified pyritide BGCs. This work exemplifies complementary bioinformatic, enzymological, and synthetic techniques to characterize a structurally distinct class of RiPP natural product.</p>

scholarly journals Structure Prediction and Synthesis of a New Class of Macrocyclic Peptide Natural Products

2020 ◽  
Author(s):  
graham a hudson ◽  
Annie R. Hooper ◽  
Adam J. DiCaprio ◽  
David Sarlah ◽  
Douglas Mitchell
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Structure Prediction ◽  
Gene Clusters ◽  
Leader Peptide ◽  
Biosynthetic Gene Clusters ◽  
New Class ◽  
Precursor Peptide ◽  
Modified Peptides ◽  
Subsequent Elimination

<p>Owing to advances in genomic sequencing and bioinformatics, the breadth of natural product biosynthetic gene clusters (BGCs) has meteorically risen. This remains true for ribosomally synthesized and post-translationally modified peptides (RiPPs), where the rate of bioinformatically identifying clusters vastly outpaces characterization efforts. Uniting bioinformatics and enzymological knowledge to predict the chemical product(s) of a RiPP BGC with total chemical synthesis to obtain the natural compound is an effective platform for investigating cryptic gene clusters. Herein, we report the bioinformatic identification of a biosynthetically divergent class of RiPP bearing a subset of enzymes involved in thiopeptide biosynthesis. These natural products were predicted based on BGC architecture to undergo a formal, enzymatic [4+2]-cycloaddition with subsequent elimination of the leader peptide and water to produce a tri-substituted pyridine-based macrocycle. Bearing a pyridine similar to thiopeptides but lacking the ubiquitous thiazole heterocycles, these new RiPPs were termed pyritides. One of the predicted natural products was chemically synthesized using an 11-step synthesis. This structure was verified to be chemically identical by an orthogonal chemoenzymatic synthesis utilizing the precursor peptide and the cognate [4+2]-cycloaddition enzyme. The chemoenzymatic platform was used to synthesize a second in-cluster pyritide product as well as analogs from other bioinformatically identified pyritide BGCs. This work exemplifies complementary bioinformatic, enzymological, and synthetic techniques to characterize a structurally distinct class of RiPP natural product.</p>

scholarly journals Identification of an Auxiliary Leader Peptide-Binding Protein Required for Azoline Formation in Ribosomal Natural Products

2015 ◽  
Vol 137 (24) ◽  
pp. 7672-7677 ◽  
Author(s):  
Kyle L. Dunbar ◽  
Jonathan I. Tietz ◽  
Courtney L. Cox ◽  
Brandon J. Burkhart ◽  
Douglas A. Mitchell
Keyword(s):  
Natural Products ◽  
Binding Protein ◽  
Peptide Binding ◽  
Leader Peptide
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