Biology subjects

van der Donk, W. A.

Publications and source records attributed to van der Donk, W. A..

14 recordsLinked to original sources

bioRxiv2026

Unconventional biocatalytic strategies orchestrate synthesis of the nucleoside analog sinefungin

Sinefungin is a potent nucleoside antimetabolite of S-adenosylmethionine (SAM), yet its biosynthesis has remained unclear for decades. Here we detail the identification and characterization of the complete sinefungin biosynthetic gene cluster (BGC) from Streptomyces incarnatus NRRL 8089. In vitro and in vivo analyses demonstrate that the defining carbon-carbon (C-C) bond is formed not by the long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme. Using isotope-labeled cofactors and substrates, we provide evidence that the adenosyl group of sinefungin atypically originates from adenosylcobalamin via a homolytic SH2 substitution, establishing a rare instance where adenosylcobalamin is enzymatically consumed during the reaction. Furthermore, the pathway utilizes a cryptic phosphorylation-dephosphorylation strategy to control intermediate processing and substrate recognition. We also characterize two peptide aminoacyl-tRNA ligases (PEARLs) that append alanines onto the nucleoside scaffold using tRNA-activated amino acids. The PEARLs act directly on small molecules rather than macromolecular substrates, with one PEARL capable of iterative elongation. Finally, we leverage these enzymes in a reduced multi-enzyme cascade to biosynthesize sinefungin. Together, these findings redefine radical-mediated C-C bond formation and pearlin enzyme versatility, unlocking biocatalytic possibilities to produce amino acid-nucleoside conjugates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/726688v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@10e48deorg.highwire.dtl.DTLVardef@d220ceorg.highwire.dtl.DTLVardef@167e60borg.highwire.dtl.DTLVardef@2fddec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Loss of LanC-like proteins impairs post-injury regeneration of aging muscles

The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced sizes of regenerating myofibers in middle-aged (but not young) LTKO compared to age-matched WT mice. Although the pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at day 5 after injury, accompanied by drastically decreased numbers of MyoD+ and MyoG+ cells, as well as increased numbers of proliferating cells. In addition, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury.

physiology↗

bioRxiv2026

Structure and Mechanism of a Two-component Lanthipeptide Toxin

The enterococcal cytolysin is a two-component lanthipeptide natural product that co-operatively kills mammalian cells and bacteria. It is a virulence factor in typically commensal strains of gut-dwelling Enterococcus faecalis and causes a fatal form of alcoholic hepatitis. Despite its clinical significance, little is known about the mechanism of action. We present here the molecular details of cytolysins remarkable bioactivity. A high-resolution cryo-electron microscopy structure revealed highly ordered tubular assemblies comprising the two subunits. We further demonstrate these structures disrupt the membranes of eukaryotic and bacterial cells. These results provide the first high-resolution structure of two distinct lanthipeptides interacting with one another and offer an explanation for the unique bioactivity of the cytolysin toxin.

biochemistry↗

bioRxiv2026

Structure, biosynthesis, and bioactivity of nostolysamides

A recent genome mining study identified class II lanthipeptides encoded in Nostoc punctiforme PCC 73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were not determined. In this study, we produced the nostolysamides by co-expression of the NpuA precursor peptide with an N-terminal SUMO tag with the class II lanthipeptide synthetase NpuM in Escherichia coli. All four lanthionine and methyllanthionine residues were shown to have the DL configuration by Marfeys analysis. Tandem mass spectrometry and mutagenesis studies indicate an N-terminal non-overlapping methyllanthionine ring and three overlapping rings at the C-terminus for which the most likely ring pattern is proposed. The NpuM lanthipeptide synthetase is a member of the ProcM-clade and catalyzes ring formation with both C-to-N and N-to-C directionality. After removal of the leader peptide, the resulting lanthipeptide exhibits antibacterial as well as antifungal activity against Candida species by disrupting cell membranes. Antibacterial activity is shown not to involve lipid II. The biosynthetic gene cluster also encodes an acetyltransferase NpuN that transfers long chain acyl groups to the side chain of a Lys residue in position 1 of the precursor peptide. In vitro studies of NpuN show relatively broad substrate specificity with NpuN conjugating various acyl groups from acyl-CoA substrates to Lys1 in the nostolysamides. The acylation did not appreciably change the antifungal and antimicrobial activity of nostolysamide showing that it is not required for these activities.

biochemistry↗

bioRxiv2026

Discovery of the Phosphonate Flavophos Produced by Burkholderia

Phosphonate natural products have proven value to society as antibiotics and herbicides. They inhibit a range of enzyme targets usually by mimicking the enzyme substrates. In this study, we investigate a family of phosphonate biosynthetic gene clusters (BGCs) found in Burkholderia. Heterologous expression in Escherichia coli resulted in production of an antimicrobial compound. Spectroscopic characterization and chemical synthesis assigned its structure as 2,4-dioxopentylphosphonic acid. One of the biosynthetic enzymes is a member of the domain of unknown function (DUF) 849 family with homology to {beta}-keto acid cleavage enzymes (BKACEs). In vitro characterization shows that this enzyme catalyzes chemistry that is divergent from previously characterized BKACEs. The observed catalytic activity is explained by a series of co-crystal structures with substrates and intermediates. The BGC also contains a gene encoding lumazine synthase (LS), an essential enzyme in flavin biosynthesis. Biochemical experiments revealed that 2,4-dioxopentylphosphonic acid inhibits LS. In addition, expression of the LS encoded in the BGC, or LS orthologs from a range of organisms, in E. coli conferred resistance to the new phosphonate, which we therefore name flavophos.

biochemistry↗

bioRxiv2025

Co-opting the bacterial lipoprotein pathway in the biosynthesis of a lipidated macrocyclic peptide

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally diverse natural products that possess a range of bioactivities, often acting as antibiotics, antifungals, or metallophores. In RiPP biosynthesis, different modifying enzymes install an array of chemical motifs onto a precursor peptide. A recently described RiPP-modifying enzyme, ChrH, catalyzes a remarkably complex reaction on its precursor peptide that results in a macrocycle, heterocycle, and S-methyl group. By leveraging comparative genomics, we demonstrate that the products from a subfamily of enzymes related to ChrH display unexpected structural diversity, including the production of unmethylated macrocyclic congeners and C-terminally modified proteins over 30 kDa in size. Several of these precursors contain a signal peptide, sending them for downstream maturation by the bacterial lipoprotein biosynthetic pathway. Like bacterial lipoproteins, such peptides are modified by addition of a diacylglycerol (DAG) group to the N-terminal cysteine residue along with acylation of the N-terminal amine. Genome mining reveals that these RiPP-lipoprotein hybrids, which we term DAG-RiPPs, are widespread across bacterial phyla and are likely involved in different biological roles. Together, these results highlight a novel maturation paradigm for membrane-bound RiPPs and lay the foundation for the discovery and bioengineering of other RiPP-lipoprotein hybrids. SignificanceRibosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of natural products that display antibiotic, antifungal, anticancer, and metal-binding activities. Their biosynthesis typically follows a common logic in which modifying enzymes install chemical motifs onto a precursor peptide, followed by proteolytic processing and export from the cell. Herein, we describe the discovery and biochemical characterization of a new class of lipid-RiPP hybrid products. These RiPPs contain a signal peptide that exploits the endogenous bacterial lipoprotein biosynthesis pathway for lipidation, membrane localization, and potential secretion. Genome mining shows that these lipid-peptide hybrids are widespread across bacterial phyla.

biochemistry↗

bioRxiv2025

An unusual ring pattern in the Rosβ lanthipeptide of the two-component lantibiotic roseocin

Two-component lantibiotics comprise two post-translationally modified peptides that synergistically exert antimicrobial activity. Most known two-component lantibiotics are made up of a structurally conserved -lanthipeptide that binds to the peptidoglycan precursor lipid II and a {beta}-lanthipeptide that is believed to interact with the -peptide-lipid II complex to form pores in the membranes of susceptible bacteria. A few two-component lantibiotics including roseocin do not follow this general scheme and act by different, currently unresolved mechanisms. An important first step in studying this group of lanthipeptides is determination of their chemical structure. Roseocins {beta}-peptide (Ros{beta}) is formed by the RosM lanthipeptide synthetase from the RosA1 precursor peptide. RosM carries out nine dehydrations of Ser and Thr residues to the corresponding dehydroamino acids followed by six Michael-type additions of the thiols of Cys residues to a subset of the dehydroamino acids forming six thioether crosslinks. Sequence alignment with structurally characterized lanthipeptides does not allow prediction of its ring pattern. In this study, Ros{beta} was produced in Escherichia coli and its ring pattern was established by multi-dimensional NMR spectroscopy. The stereochemistry of the lanthionine and methyllanthionine residues was determined by Marfeys analysis with authentic standards. Ros{beta} is shown to have a unique ring pattern amongst previously characterized lanthipeptides. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/684949v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@125226borg.highwire.dtl.DTLVardef@f6a170org.highwire.dtl.DTLVardef@1e0e57corg.highwire.dtl.DTLVardef@1fa4c8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Aminoacyl-tRNA specificity of a ligase catalyzing non-ribosomal peptide extension

Peptide aminoacyl-transfer ribonucleic acid ligases (PEARLs) are amide bond-forming enzymes that extend the main chain of peptides using aminoacyl-tRNA (aa-tRNA) as a substrate. In this study, we investigated the substrate specificity of the PEARL BhaBCAla from Bacillus halodurans, which utilizes Ala-tRNAAla. By leveraging flexizyme, a ribozyme capable of charging diverse acids onto a desired tRNA, we generated an array of aa-tRNAs in which we varied both the amino acid and the tRNA to dissect the substrate scope of BhaBCAla. We demonstrate that BhaBCAla catalyzes peptide extension with non-cognate proteinogenic and non-canonical amino acids, hydroxy acids, and mercaptocarboxylic acids when attached to tRNAAla. For most of these, the efficiency was considerably reduced compared to Ala, indicating the enzyme recognizes the amino acid. By varying the different parts of the tRNA, enzyme specificity was shown to also depend on the acceptor stem and the anticodon arm of the tRNA. These findings establish the molecular determinants of PEARL specificity and provide a foundation for engineering these enzymes for broader applications in peptide synthesis.

biochemistry↗

bioRxiv2025

The Biosynthetic Pathway to the Pyrroloiminoquinone Marine Natural Product Ammosamide C

Ammosamide C is a marine natural product containing a highly decorated pyrroloiminoquinone core. Studies on the biosynthetic gene cluster (BGC) that produces ammosamides previously revealed that they are made by a series of posttranslational modifications (PTMs). The BGC includes genes encoding a precursor peptide AmmA and four enzymes known as PEptide Aminoacyl-tRNA Ligases (PEARLs). Initial studies into the ammosamide biosynthetic pathway demonstrated Trp addition to a precursor peptide by the PEARL AmmB2. Thereafter, sequential modifications by several enzymes including two other PEARLs lead to the formation of a peptide intermediate bearing a C-terminal diaminoquinone. In the present work, we present the biosynthetic steps that convert this intermediate to ammosamide C. The PEARL AmmB4 unexpectedly appends an arginine to the C-terminus of the aforementioned intermediate. Then, C-terminal proteolysis by the heterodimeric TldD/E-like protease Amm12/13 releases a dipeptide, which is subsequently cleaved by the dipeptidase Amm19 to produce a Trp-derived diaminoquinone. Amm3 next catalyzes the conversion of this Trp derivative to the corresponding chlorinated ammosamaic acid. Finally, Amm23 methylates this intermediate and a putative aminotransferase Amm20 performs an amidation to arrive at ammosamide C; the order of these last two steps could not be determined. This study reveals an unexpectedly lengthy route to ammosamide that illustrates the opportunistic nature of natural product biosynthesis, demonstrates a role for a PEARL that is unlike previous roles, identifies steps that are not PTMs, and adds Arg-tRNA to the growing repertoire of amino acyl tRNAs that are used by PEARLs.

biochemistry↗

bioRxiv2025

Biosynthesis of Macrocyclic Peptides by Formation and Crosslinking of ortho-Tyrosines

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a growing class of natural products that possess many activities that are of potential translational interest. Multinuclear non-heme iron dependent oxidative enzymes (MNIOs), until recently termed domain of unknown function 692 (DUF692), have been gaining interest because of their involvement in a range of biochemical reactions that are remarkable from a chemical perspective. Over 13,500 putative MNIO-encoding biosynthetic gene clusters (BGCs) have been identified by sequence similarity networks (SSNs). In this study, we identified a set of precursor peptides containing a conserved FHAFRF-motif in MNIO-encoding BGCs. These BGCs follow a conserved synteny with genes encoding an MNIO, a RiPP recognition element (RRE)-containing partner protein, an arginase, and a B12-dependent radical SAM enzyme (rSAM). Using heterologous reconstitution of a representative BGC from Peribacillus simplex (pbs cluster) in E. coli, we demonstrated that the MNIO in conjunction with the partner protein catalyzes ortho-hydroxylation of each of the phenylalanine residues in the conserved FRF-motif, the arginase forms an ornithine by deguanidination of the arginine in the motif, and the B12-rSAM crosslinks the ortho-Tyr side side chains by a C-C linkage forming a novel macrocyclic molecule. Substrate scope studies suggested tolerance of the MNIO and the B12-rSAM towards substituting the Phe residues with tyrosines in the conserved motif with the position of hydroxylation and crosslinking being maintained. Overall, this study expands the diverse array of posttranslational modifications catalyzed by MNIOs and B12-rSAM enzymes. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/647296v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@37d9c8org.highwire.dtl.DTLVardef@baf547org.highwire.dtl.DTLVardef@3d0ed8org.highwire.dtl.DTLVardef@99aaee_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Kinetic Analysis of Cyclization Reactions Performed by Substrate-Tolerant ProcM

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of thioether crosslinks. Class II lanthipeptide synthetases are bifunctional enzymes responsible for the multistep chemical modification of these natural products. ProcM is a class II lanthipeptide synthetase known for its remarkable substrate tolerance and ability to install diverse (methyl)lanthionine rings with high accuracy. Previous studies suggested that the final ring pattern of the lanthipeptide product may be determined by the substrate sequence rather than by ProcM, and that ProcM operates by a kinetically controlled mechanism, wherein the ring pattern is dictated by the relative rates of the individual cyclization reactions. This study utilizes kinetic assays to determine if rates of isolated modifications can predict the final ring pattern present in prochlorosins. Changes in the core substrate sequence resulted in changes to the reaction rates of ring formation as well as a change in the order of modifications. Additionally, individual chemical reaction rates were significantly impacted by the presence of other modifications on the peptide. These findings indicate that the rates of isolated modifications are capable of predicting the final ring pattern but are not necessarily a good predictor of the order of modification in WT ProcA3.3 and its variants.

biochemistry↗

bioRxiv2023

Structure-Activity Relationship Studies of Novel Gut-derived Lantibiotics Against Human Gut Commensals

Recent advances in sequencing techniques unveiled the vast potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) encoded in microbiomes. Class I lantibiotics such as nisin A, widely used as a food preservative, have been investigated for their efficacy in killing pathogens. However, the impact of nisin and nisin-like class I lantibiotics on commensal bacteria residing in the human gut remains unclear. Here, we report six gut-derived class I lantibiotics that are close homologs of nisin, four of which are novel. We applied an improved lantibiotic expression platform to produce and purify these lantibiotics for antimicrobial assays. We determined their minimal inhibitory concentration (MIC) against both Gram-positive human pathogens and gut commensals, and profiled the lantibiotic resistance genes in these pathogens and commensals. SAR studies with variants revealed key regions and residues that impact their antimicrobial properties. Our characterization and SAR studies of nisin-like lantibiotics against both pathogens and human gut commensals could shed light on the future development of lantibiotic-based therapeutics and food preservatives.

bioengineering↗

bioRxiv2023

A remarkable transformation catalyzed by a domain-of-unknown-function 692 during the biosynthesis of a new RiPP natural product

The domain of unknown function 692 (DUF692) is an emerging family of posttranslational modification enzymes involved in the biosynthesis of ribosomally-synthesized and posttranslationally modified peptide (RiPP) natural products. Members of this family are multinuclear iron-containing enzymes and only two members have been functionally characterized to date: MbnB and TglH. Here, we used bioinformatics to select another member of the DUF692 family, ChrH, that is ubiquitously encoded in the genomes of the Chryseobacterium genus along with a partner protein ChrI. We structurally characterized the ChrH reaction product and show that the enzyme catalyzes an unprecedented chemical transformation that results in the formation of a macrocycle, an imidazolidinedione heterocycle, two thioaminals, and a thiomethylation. Based on isotopic labeling studies, we propose a mechanism for the four-electron oxidation and methylation of the substrate peptide. This work identifies the first SAM-dependent DUF692 enzyme, further expanding the repertoire of remarkable reactions catalyzed by these enzymes.

biochemistry↗

bioRxiv2020

Precursor peptide-targeted mining of more than one hundred thousand genomes expands the lanthipeptide natural product family

BackgroundLanthipeptides belong to the ribosomally synthesized and post-translationally modified peptide group of natural products and have a variety of biological activities ranging from antibiotics to antinociceptives. These peptides are cyclized through thioether crosslinks and can bear other secondary post-translational modifications. While lanthipeptide biosynthetic gene clusters can be identified by the presence of characteristic enzymes involved in the post-translational modification of these peptides, locating the precursor peptides encoded within these clusters is challenging due to their short length and high sequence variability, which limits the high-throughput exploration of lanthipeptide precursor peptides. To address this challenge, we enhanced the predictive capabilities of Rapid ORF Description & Evaluation Online (RODEO) to identify all known classes of lanthipeptides. ResultsUsing RODEO, we mined over 100,000 bacterial and archaeal genomes in the RefSeq database. We identified nearly 8,500 lanthipeptide precursor peptides. These precursor peptides were identified in a broad range of bacterial phyla as well as the Euryarchaeota phylum of archaea. Bacteroidetes were found to encode a large number of these biosynthetic gene clusters, despite making up a relatively small portion of the genomes in this dataset. While a number of these precursor peptides are similar to those of previously characterized lanthipeptides, even more were not, including potential antibiotics. Additionally, examination of the biosynthetic gene clusters revealed enzymes that install secondary post-translational modifications are more widespread than initially thought. ConclusionLanthipeptide biosynthetic gene clusters are more widely distributed and the precursor peptides encoded within these clusters are more diverse than previously appreciated, demonstrating that the lanthipeptide sequence-function space remains largely underexplored.

bioinformatics↗