1,578 research outputs found
Cytochromes P450 involved in bacterial RiPP biosyntheses
No full textRibosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of secondary metabolites that have garnered scientific attention due to their complex scaffolds with potential roles in medicine, agriculture, and chemical ecology. RiPPs derive from the cleavage of ribosomally synthesized proteins and additional modifications, catalyzed by various enzymes to alter the peptide backbone or side chains. Of these enzymes, cytochromes P450 (P450s) are a superfamily of heme-thiolate proteins involved in many metabolic pathways, including RiPP biosyntheses. In this review, we focus our discussion on P450 involved in RiPP pathways and the unique chemical transformations they mediate. Previous studies have revealed a wealth of P450s distributed across all domains of life. While the number of characterized P450s involved in RiPP biosyntheses is relatively small, they catalyze various enzymatic reactions such as C-C or C-N bond formation. Formation of some RiPPs is catalyzed by more than one P450, enabling structural diversity. With the continuous improvement of the bioinformatic tools for RiPP prediction and advancement in synthetic biology techniques, it is expected that further cytochrome P450-mediated RiPP biosynthetic pathways will be discovered.
SUMMARY: The presence of genes encoding P450s in gene clusters for ribosomally synthesized and post-translationally modified peptides expand structural and functional diversity of these secondary metabolites, and here, we review the current state of this knowledge
NeuRiPP : neural network identification of RiPP precursor peptides
No full textSignificant progress has been made in the past few years on the computational identification of biosynthetic gene clusters (BGCs) that encode ribosomally synthesized and post-translationally modified peptides (RiPPs). This is done by identifying both RiPP tailoring enzymes (RTEs) and RiPP precursor peptides (PPs). However, identification of PPs, particularly for novel RiPP classes remains challenging. To address this, machine learning has been used to accurately identify PP sequences. Current machine learning tools have limitations, since they are specific to the RiPPclass they are trained for and are context-dependent, requiring information about the surrounding genetic environment of the putative PP sequences. NeuRiPP overcomes these limitations. It does this by leveraging the rich data set of high-confidence putative PP sequences from existing programs, along with experimentally verified PPs from RiPP databases. NeuRiPP uses neural network archictectures that are suitable for peptide classification with weights trained on PP datasets. It is able to identify known PP sequences, and sequences that are likely PPs. When tested on existing RiPP BGC datasets, NeuRiPP was able to identify PP sequences in significantly more putative RiPP clusters than current tools while maintaining the same HMM hit accuracy. Finally, NeuRiPP was able to successfully identify PP sequences from novel RiPP classes that were recently characterized experimentally, highlighting its utility in complementing existing bioinformatics tools
Expansion of predicted RiPP biosynthetic sequence space using the RiPP recognition element
No full textSubmission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2025-08-01The student, Kyle Shelton, accepted the attached license on 2023-07-03 at 17:50.The student, Kyle Shelton, submitted this Dissertation for approval on 2023-07-03 at 18:01.This Dissertation was approved for publication on 2023-07-13 at 20:30.DSpace SAF Submission Ingestion Package generated from Vireo submission #19506 on 2023-12-04 at 17:30:43Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products for which discovery efforts have rapidly grown over the past decade. More than half of the prokaryotic RiPP classes include a protein domain called the RiPP Recognition Element (RRE) for successful installation of post-translational modifications on a RiPP precursor peptide. In most cases, the RRE domain binds to the N-terminal ‘leader’ region of the precursor peptide, facilitating enzymatic modification of the C-terminal ‘core’ region. The prevalence of the RRE domain renders it a theoretically useful bioinformatic handle for class independent RiPP discovery. Moreover, with most known RRE domains engaging their cognate precursor peptide(s) with high specificity and nanomolar affinity, evaluation of the residue-specific interactions that govern RRE:substrate complexation is a necessary first step to leveraging the RRE domain for various bioengineering applications. Chapter 1 details protocols for developing custom bioinformatic models to predict and annotate RRE domains in a class-specific manner. Methods are outlined for experimental validation of precursor peptide binding using fluorescence polarization binding assays and in vitro enzyme activity assays. Chapter 2 presents a novel genome mining tool, RRE-Finder, created to identify and annotate RRE domains from genomic data. Given its prevalence across various types of RiPP biosynthetic gene clusters (BGCs), the RRE can be used as a bioinformatic handle to identify novel classes of RiPPs. However, sequence divergence of RREs across RiPP classes has precluded automated identification based solely on sequence similarity. RRE-Finder is a tool for identifying RRE domains with high sensitivity. RRE-Finder can be used in “precision” mode to confidently identify RREs in a class-specific manner or in “exploratory” mode, to assist in the discovery of novel RiPP classes. RRE-Finder operating in precision mode on the UniProtKB protein database retrieved ~25,000 high-confidence RREs spanning all characterized RRE-dependent RiPP classes, as well as several yet-uncharacterized RiPP classes that require future experimental confirmation. Finally, RRE-Finder was used in precision mode to explore a possible evolutionary origin of the RRE domain. The results suggest RREs originated from a co-opted DNA-binding transcriptional regulator domain. Altogether, RRE-Finder provides a powerful new method to probe RiPP biosynthetic diversity and delivers a rich dataset of RRE sequences that will provide a foundation for deeper biochemical studies into this intriguing and versatile protein domain. Chapter 3 leverages RRE-Finder precision mode datasets to delve into the relationship between RRE domains and their cognate precursor peptides. The identification of recognition sequences in RiPP precursor peptides is important not only from the perspective of accurately predicting viable precursors in genomic contigs but also for leveraging possible biotechnological applications of the RRE in protein purification and detection. In this chapter, we bioinformatically survey the landscape of RS motifs found across all RRE-dependent RiPP classes, highlighting 26 widespread RS motifs that are prevalent in one or more RiPP classes. Next, we employ in vitro binding assays to quantify binding interactions for several as-of-yet uncharacterized RRE:RS interactions, using this binding data to validate or disprove hypotheses generated by AlphaFold models for critical RS residues in these complexes
Conformational rearrangements enable iterative backbone -methylation in RiPP biosynthesis
No full textR Code:
Supplementary Data 1. Code for running kinetics simulations
File name: Supplementary Data 1.R
Mass Spectrometry .raw files:
Supplementary Figure 4a-c. Mass spectrometric analysis of split borosin coexpressions.
File name: 20200110_FSM1167_SonA+SonM_24.5min.raw
Supplementary Figure 4d-j. Mass spectrometric analysis of split borosin coexpressions.
File name: 20200909_KC1063_StrA+StrM_15min_redalk_18pm4.raw
Supplementary Figure 16. SonM in vitro reactions analyzed by LC-MS/MS and
compared to kinetic model simulations.
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Supplementary Figure 25a. Mass spectrometric analysis of SonM mutant in vitro
reactions.
File name: 20190701_kc1007_His-SonA_Y93F.raw
Supplementary Figure 25b. Mass spectrometric analysis of SonM mutant in vitro
reactions.
File name: 20190515_fsm1155_his-SonA-sonMT-R67K.raw
Supplementary Figure 25c. Mass spectrometric analysis of SonM mutant in vitro
reactions.
File name: 20190515_fsm1154_his-sonA_sonMTR67A.raw
Supplementary Figure 25d. Mass spectrometric analysis of SonM mutant in vitro
reactions.
File name: 20190701_kc1008_His-SonA_Y58F.raw
Supplementary Figure 25e. Mass spectrometric analysis of SonM mutant in vitro
reactions.
File name: 20190515_fsm1156_his-sonA-SonMT-Y71F.raw
Supplementary Figure 25f. Mass spectrometric analysis of SonM mutant in vitro
reactions.
File name: 20190701_kc1009_His-SonA_DBLMUT.rawThe data deposited here are raw files, R code and mass spec, associated with the results presented in the paper, "Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis." These data have been made publicly available in keeping with the journal's data availability policy.National Institutes of Health (R35 GM133475 to M.F.F.) and the University of Minnesota along with the BioTechnology Institute (M.F.F., M.H.E., W.A.H.)Miller, Fredarla S.; Crone, Kathryn K.; Jensen, Matthew R.; Shaw, Sudipta; Harcombe, William R.; Elias, Mikael H.; Freeman, Michael F.. (2021). Conformational rearrangements enable iterative backbone -methylation in RiPP biosynthesis. Retrieved from the University Digital Conservancy, https://doi.org/10.13020/y8ry-gm18
Disordered regions in proteusin peptides guide post-translational modification by a flavin-dependent RiPP brominase
No full textAbstract To biosynthesize ribosomally synthesized and post-translationally modified peptides (RiPPs), enzymes recognize and bind to the N-terminal leader region of substrate peptides which enables catalytic modification of the C-terminal core. Our current understanding of RiPP leaders is that they are short and largely unstructured. Proteusins are RiPP precursor peptides that defy this characterization as they possess unusually long leaders. Proteusin peptides have not been structurally characterized, and we possess scant understanding of how these atypical leaders engage with modifying enzymes. Here, we determine the structure of a proteusin peptide which shows that unlike other RiPP leaders, proteusin leaders are preorganized into a rigidly structured region and a smaller intrinsically disordered region. With residue level resolution gained from NMR titration experiments, the intermolecular peptide-protein interactions between proteusin leaders and a flavin-dependent brominase are mapped onto the disordered region, leaving the rigidly structured region of the proteusin leader to be functionally dispensable. Spectroscopic observations are biochemically validated to identify a binding motif in proteusin peptides that is conserved among other RiPP leaders as well. This study provides a structural characterization of the proteusin peptides and extends the paradigm of RiPP modification enzymes using not only unstructured peptides, but also structured proteins as substrates
N–Cα Bond Cleavage Catalyzed by a Multinuclear Iron Oxygenase from a Divergent Methanobactin-like RiPP Gene Cluster
No full textDUF692 multinuclear iron oxygenases (MNIOs) are an emerging family of tailoring enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Three members, MbnB, TglH, and ChrH, have been characterized to date and shown to catalyze unusual and complex transformations. Using a co-occurrence-based bioinformatic search strategy, we recently generated a sequence similarity network of MNIO-RiPP operons that encode one or more MNIOs adjacent to a transporter. The network revealed >1,000 unique gene clusters, evidence of an unexplored biosynthetic landscape. In this work, we assess an MNIO-RiPP cluster from the network that is encoded in proteobacteria and actinobacteria. The cluster, which we have termed mov (for methanobactin-like operon in Vibrio), encodes a 23-residue precursor peptide, two MNIOs, a RiPP recognition element, and a transporter. Using both in vivo and in vitro methods, we show that one MNIO, homologous to MbnB, installs an oxazolone-thioamide at a Thr-Cys dyad in the precursor. Subsequently, the second MNIO catalyzes N–Cα bond cleavage of the penultimate Asn to generate a C-terminally amidated peptide. This transformation expands the reaction scope of the enzyme family, marks the first ex-ample of an MNIO-catalyzed modification that does not involve Cys, and sets the stage for future exploration of other MNIO-RiPP
Expanding the Landscape of Noncanonical Amino Acids in RiPP Biosynthesis
No full textAdvancements in DNA sequencing technologies and bioinformatics
have enabled the discovery of new metabolic reactions from overlooked
microbial species and metagenomic sequences. Using a bioinformatic
co-occurrence strategy, we previously generated a network of ∼600
uncharacterized quorum-sensing-regulated biosynthetic gene clusters
that code for ribosomally synthesized and post-translationally modified
peptide (RiPP) natural products and are tailored by radical S-adenosylmethionine (RaS) enzymes in streptococci. The
most complex of these is the GRC subfamily, named
after a conserved motif in the precursor peptide and found exclusively
in Streptococcus pneumoniae, the causative agent
of bacterial pneumonia. In this study, using both in vivo and in vitro approaches, we have elucidated the
modifications installed by the grc biosynthetic enzymes,
including a ThiF-like adenylyltransferase/cyclase that generates a
C-terminal Glu-to-Cys thiolactone macrocycle, and two RaS enzymes,
which selectively epimerize the β-carbon of threonine and desaturate
histidine to generate the first instances of l-allo-Thr and didehydrohistidine in RiPP biosynthesis. RaS-RiPPs that
have been discovered thus far have stood out for their exotic macrocycles.
The product of the grc cluster breaks this trend
by generating two noncanonical residues rather than an unusual macrocycle
in the peptide substrate. These modifications expand the landscape
of nonproteinogenic amino acids in RiPP natural product biosynthesis
and motivate downstream biocatalytic applications of the corresponding
enzymes
Nachweis eines RiPP-Precursors auf Peptid-Ebene
No full textRibosomally synthesized and post-translationally modified peptides (RiPPs) are a group of secondary metabolites with unique biosynthetic pathways resulting in a wide variety of structures and bioactivities. RiPP biosynthesis is a two-stage process. First, linear precursor peptides consisting of an N-terminal leader sequence, a core sequence and an optional C-terminal recognition sequence are translated by classical ribosomal translation. After this, the linear precursor is heavily modified by an assembly of tailoring enzymes yielding the mature, often cyclized product. A set of potential RiPP precursors has been identified in the genome of the fungus Trichoderma reesei in a recent genome mining approach and gene cluster activity was demonstrated on the transcriptome level. The aim of this thesis was to determine whether the precursor/leader sequence mRNA is translated into the corresponding precursor/leader peptides. To this end, extracts of wild type and RiPP knockout strains of T. reesei were subjected to quantitative LCIMS-MS/MS analysis. Different protease digestion strategies were tested to maximize sequence coverage of the RiPP precursor. By employing a simple extraction procedure followed by nanoLC-IMS-MS/MS analysis and a common proteomics database search, the translation of the precursor mRNA into the corresponding peptide could be verified. Moreover, several extraction and analysis approaches were tested on model RiPPs produced in the fungus Aspergillus flavus to develop an efficient method for the analysis of mature RiPPs which will set a basis for the discovery and identification of RiPPs with yet unknown structure in T. reesei. The second part of this thesis compares the efficiency of different sample preparation methods for proteomics with a special focus on sub-microgram sample input. A recently published paramagnetic bead-based approach (single-pot, solid-phase enhanced sample preparation, SP3) for sample preparation was compared to an established in-solution digestion method and a commercially available kit for sample preparation (PreOmics). The SP3 approach was further optimized by adopting different peptide cleanup strategies in order to increase the number of identified proteins. It was shown that for protein amounts above 10 μg, an increase in protein identifications can be achieved by employing an additional desalting step
Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine–Tryptophan Crosslink
No full textRibosomally
synthesized and post-translationally modified peptides
(RiPPs) are a growing family of natural products with diverse activities
and structures. RiPP classes are defined by the tailoring enzyme,
which can introduce a narrow range of modifications or a diverse set
of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs,
have emerged as especially divergent. A map of all RaS-RiPP gene clusters
does not yet exist. Moreover, precursor peptides remain difficult
to predict using computational methods. Herein, we have addressed
these challenges and report a bioinformatic atlas of RaS-RiPP gene
clusters in available microbial genome sequences. Using co-occurrence
of RaS enzymes and transporters from varied families as a bioinformatic
hook in conjunction with an in-house code to identify precursor peptides,
we generated a map of ∼15,500 RaS-RiPP gene clusters, which
reveal a remarkable diversity of syntenies pointing to a tremendous
range of enzymatic and natural product chemistries that remain to
be explored. To assess its utility, we examined one family of gene
clusters encoding a YcaO enzyme and a RaS enzyme. We find the former
is noncanonical, contains an iron–sulfur cluster, and installs
a novel modification, a backbone amidine into the precursor peptide.
The RaS enzyme was also found to install a new modification, a C–C
crosslink between the unactivated terminal δ-methyl group of
Ile and a Trp side chain. The co-occurrence search can be applied
to other families of RiPPs, as we demonstrate with the emerging DUF692
di-iron enzyme superfamily
Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine–Tryptophan Crosslink
No full textRibosomally
synthesized and post-translationally modified peptides
(RiPPs) are a growing family of natural products with diverse activities
and structures. RiPP classes are defined by the tailoring enzyme,
which can introduce a narrow range of modifications or a diverse set
of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs,
have emerged as especially divergent. A map of all RaS-RiPP gene clusters
does not yet exist. Moreover, precursor peptides remain difficult
to predict using computational methods. Herein, we have addressed
these challenges and report a bioinformatic atlas of RaS-RiPP gene
clusters in available microbial genome sequences. Using co-occurrence
of RaS enzymes and transporters from varied families as a bioinformatic
hook in conjunction with an in-house code to identify precursor peptides,
we generated a map of ∼15,500 RaS-RiPP gene clusters, which
reveal a remarkable diversity of syntenies pointing to a tremendous
range of enzymatic and natural product chemistries that remain to
be explored. To assess its utility, we examined one family of gene
clusters encoding a YcaO enzyme and a RaS enzyme. We find the former
is noncanonical, contains an iron–sulfur cluster, and installs
a novel modification, a backbone amidine into the precursor peptide.
The RaS enzyme was also found to install a new modification, a C–C
crosslink between the unactivated terminal δ-methyl group of
Ile and a Trp side chain. The co-occurrence search can be applied
to other families of RiPPs, as we demonstrate with the emerging DUF692
di-iron enzyme superfamily
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