-
Biochemistry Feb 2023The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP)...
The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded β-sheet to form a hydrophobic β-sandwich-like interface. The bioinformatics-guided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain-domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.
Topics: Protein Sorting Signals; Peptide Hydrolases; Protein Processing, Post-Translational; Bacterial Proteins; Peptides
PubMed: 36734655
DOI: 10.1021/acs.biochem.2c00700 -
Applied Microbiology and Biotechnology Aug 2012A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are... (Review)
Review
A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.
Topics: Antifungal Agents; Bacillus cereus; Bacillus thuringiensis; Lipopeptides; Molecular Structure; Peptide Biosynthesis, Nucleic Acid-Independent; Protein Conformation
PubMed: 22678024
DOI: 10.1007/s00253-012-4181-2 -
The Journal of General and Applied... Feb 2021Acrocarpospora is a rare, recently established actinomycete genus of the family Streptosporangiaceae. In the present study, we sequenced whole genomes of the type...
Acrocarpospora is a rare, recently established actinomycete genus of the family Streptosporangiaceae. In the present study, we sequenced whole genomes of the type strains of Acrocarpospora corrugate, Acrocarpospora macrocephala, and Acrocarpospora pleiomorpha to assess their potency as secondary metabolite producers; we then surveyed their nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters. The genome sizes of A. corrugate NBRC 13972, A. macrocephala NBRC 16266, and A. pleiomorpha NBRC 16267 were 9.3 Mb, 12.1 Mb, and 11.8 Mb, respectively. Each genome contained 12-17 modular NRPS and PKS gene clusters. Among the 23 kinds of NRPS and PKS gene clusters identified from the three strains, eight clusters were conserved in all the strains, six were shared between A. macrocephala and A. pleiomorpha, and the remaining nine were strain-specific. We predicted the chemical structures of the products synthesized by these gene clusters based on bioinformatic analyses. Since the chemical structures are diverse, Acrocarpospora strains are considered an attractive source of diverse nonribosomal peptide and polyketide compounds.
Topics: Actinobacteria; Base Sequence; Genome, Bacterial; Multigene Family; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Polyketide Synthases; Sequence Analysis, DNA; Whole Genome Sequencing
PubMed: 32801283
DOI: 10.2323/jgam.2020.01.001 -
Microbiology (Reading, England) May 2007In fungi, nonribosomal peptide synthetases (NRP synthetases) are large multi-functional enzymes containing adenylation, thiolation (or peptidyl carrier protein, PCP) and... (Review)
Review
In fungi, nonribosomal peptide synthetases (NRP synthetases) are large multi-functional enzymes containing adenylation, thiolation (or peptidyl carrier protein, PCP) and condensation domains. These enzymes are often encoded within gene clusters. Multiple NRP synthetase ORFs have also been identified in fungi (14 in Aspergillus fumigatus). LeaA, a methyltransferase, is involved in secondary metabolite gene cluster regulation in Aspergillus spp. The NRP synthetases GliP and FtmA respectively direct the biosynthesis of the toxic metabolites gliotoxin and brevianamide F, a precursor of bioactive prenylated alkaloids. The NRP synthetase Pes1 has been shown to mediate resistance to oxidative stress, and in plant-pathogenic ascomycetes (e.g. Cochliobolus heterostrophus) an NRP synthetase, encoded by the NPS6 gene, significantly contributes to virulence and resistance to oxidative stress. Adenylation (A) domains within NRP synthetases govern the specificity of amino acid incorporation into nonribosomally synthesized peptides. To date there have only been limited demonstrations of A domain specificity (e.g. A. fumigatus GliP and in Beauveria bassiana) in fungi. Indeed, only in silico prediction data are available on A domain specificity of NRP synthetases from most fungi. NRP synthetases are activated by 4'-phosphopantetheinylation of serine residues within PCP domains by 4'-phosphopantetheinyl transferases (4'-PPTases). Coenzyme A acts as the 4'-phosphopantetheine donor, and labelled coenzyme A can be used to affinity-label apo-NRP synthetases. Emerging fungal gene disruption and gene cluster expression strategies, allied to proteomic strategies, are poised to facilitate a greater understanding of the coding potential of NRP synthetases in fungi.
Topics: Aspergillus fumigatus; Fungi; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases
PubMed: 17464044
DOI: 10.1099/mic.0.2006/006908-0 -
Fungal Genetics and Biology : FG & B Jan 2011With many bioactive non-ribosomal peptides and polyketides produced in fungi, studies of their biosyntheses are an active area of research. Practical limitations of... (Review)
Review
With many bioactive non-ribosomal peptides and polyketides produced in fungi, studies of their biosyntheses are an active area of research. Practical limitations of working with mega-dalton synthetases including cell lysis and protein extraction to recombinant gene and pathway expression has slowed understanding of many secondary metabolic processes relative to bacterial counterparts. Recent advances in accessing fungal biosynthetic machinery are beginning to change this. Here we describe the successes of some studies of thiotemplate biosynthesis in fungal systems, along with very recent advances in chemical tagging and mass spectrometric strategies to selectively study biosynthetic conveyer belts in isolation, and within a few years, in endogenous fungal proteomes.
Topics: Fungi; Macrolides; Mass Spectrometry; Peptide Biosynthesis, Nucleic Acid-Independent; Peptides; Proteomics
PubMed: 20601041
DOI: 10.1016/j.fgb.2010.06.012 -
Cell Apr 2022Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies...
Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies dating back to the 1920s show that female schistosomes rely on constant physical contact with a male worm both to become and remain sexually mature; however, the molecular details governing this process remain elusive. Here, we uncover a nonribosomal peptide synthetase that is induced in male worms upon pairing with a female and find that it is essential for the ability of male worms to stimulate female development. We demonstrate that this enzyme generates β-alanyl-tryptamine that is released by paired male worms. Furthermore, synthetic β-alanyl-tryptamine can replace male worms to stimulate female sexual development and egg laying. These data reveal that peptide-based pheromone signaling controls female schistosome sexual maturation, suggesting avenues for therapeutic intervention and uncovering a role for nonribosomal peptides as metazoan signaling molecules.
Topics: Animals; Female; Male; Peptide Biosynthesis, Nucleic Acid-Independent; Peptides; Pheromones; Schistosoma; Tryptamines
PubMed: 35385687
DOI: 10.1016/j.cell.2022.03.017 -
Current Opinion in Chemical Biology Aug 2012Redox enzymes such as FAD-dependent and cytochrome P450 oxygenases play indispensible roles in generating structural complexity during natural product biosynthesis. In... (Review)
Review
Redox enzymes such as FAD-dependent and cytochrome P450 oxygenases play indispensible roles in generating structural complexity during natural product biosynthesis. In the pre-assembly steps, redox enzymes can convert garden variety primary metabolites into unique starter and extender building blocks. In the post-assembly tailoring steps, redox cascades can transform nascent scaffolds into structurally complex final products. In this review, we will discuss several recently characterized redox enzymes in the biosynthesis of polyketides and nonribosomal peptides.
Topics: Biological Products; Enzymes; Oxidation-Reduction; Peptide Biosynthesis, Nucleic Acid-Independent; Polyketides
PubMed: 22564679
DOI: 10.1016/j.cbpa.2012.04.008 -
Journal of Molecular Biology May 2016In order to colonize a niche and compete for scarce resources, microorganisms have evolved means to adjust the expression levels of their biosynthetic enzymes in... (Review)
Review
In order to colonize a niche and compete for scarce resources, microorganisms have evolved means to adjust the expression levels of their biosynthetic enzymes in response to the changing levels of metabolites available to them. To do so, they often rely on transcription factors or structured RNAs that directly sense the concentration of metabolites and turn genes on or off accordingly. In some instances, however, a metabolite can be sensed by an actively translating ribosome bearing a nascent polypeptide whose specific amino acid sequence interferes with translation. These “arrest peptides” lead to the formation of stalled ribosome nascent chain complexes on the mRNA that can regulate the expression of downstream genes through transcriptional or translational mechanisms. Although this process was discovered over three and a half decades ago, the extent to which arrest peptides regulate gene expression in response to cell metabolites is unknown. Here, we examine the physical constraints imposed by the ribosome on peptide-mediated ligand sensing and review attempts to assess the diversity of arrest peptides to date. In addition, we outline a possible way forward to establish how pervasive metabolite sensing by arrest peptides is in nature.
Topics: Gene Expression Regulation, Bacterial; Models, Biological; Peptide Biosynthesis; Protein Biosynthesis; Ribosomes
PubMed: 27108680
DOI: 10.1016/j.jmb.2016.04.019 -
The Journal of Biological Chemistry Oct 2019The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are...
The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen By combining and approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti- peptide RumC depends on an operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.
Topics: Amino Acid Sequence; Bacteriocins; Clostridiales; Clostridium perfringens; Gastrointestinal Microbiome; Humans; Multigene Family; Peptide Biosynthesis; Peptides; Protein Processing, Post-Translational; Ribosomes; Sterile Alpha Motif; Sulfides; Symbiosis
PubMed: 31337708
DOI: 10.1074/jbc.RA119.009416 -
FEBS Letters Jul 1992Biosynthesis of peptides in non-ribosomal systems is catalyzed by multifunctional enzymes that employ the thio-template mechanism. Recent studies on the analysis of the... (Review)
Review
Biosynthesis of peptides in non-ribosomal systems is catalyzed by multifunctional enzymes that employ the thio-template mechanism. Recent studies on the analysis of the primary structure of several peptide synthetases have revealed that they are organized in highly conserved and repeated functional domains. The aligned domains provide the template for peptide synthesis, and their order determines the sequence of the peptide product.
Topics: Amino Acid Sequence; Molecular Sequence Data; Multienzyme Complexes; Peptide Biosynthesis; Peptide Synthases
PubMed: 1639192
DOI: 10.1016/0014-5793(92)80898-q