Biology subjects

Link, A. J.

Publications and source records attributed to Link, A. J..

11 recordsLinked to original sources

bioRxiv2025

Structural insights into xyloglucan recognition by an ABC transporter from a Gram-positive, thermophilic bacterium

Xyloglucan (an -1,6-xylosyl-substituted {beta}-1,4-glucan) is a major hemicellulose of the primary cell wall of many plants and an important growth substrate for biomass-degrading bacteria in diverse ecological niches, including the gut microbiome and hot springs. In Gram-positive bacteria, xyloglucan is deconstructed into soluble oligosaccharides in the extracytoplasmic space before import by ATP-Binding Cassette (ABC) transporters, but the structural basis for this process remains poorly understood. Here, we identified an ABC transporter for xyloglucan uptake (Athe_2052-2054) in the Gram-positive, plant biomass-degrading thermophile Anaerocellum bescii, which is conserved across the Anaerocellum genus. We solved the apo crystal structure of its extracellular substrate-binding protein (SBP), Athe_2052, revealing a unique tertiary fold found only in a small subset of SBPs that bind complex oligosaccharides. This structure represents the first ABC SBP known to bind xyloglucan oligosaccharides. Biophysical analysis showed that while Athe_2052 binds unsubstituted {beta}-glucan chains, recognition of xyloglucan side chains in the binding pocket markedly increases affinity (Kd = 14 nM) for xyloglucan heptasaccharide (XXXG), the principal oligosaccharide released during xyloglucan deconstruction. Molecular modeling revealed that xyloglucan heptasaccharide, owing to its branched substitutions, is bound in a distinct conformation compared to unsubstituted {beta}-glucans. This represents a unique mode of xyloglucan recognition driven by -linked side-chain interactions rather than {beta}-glucan backbone recognition alone. Together, these findings provide the first structural basis for xyloglucan oligosaccharide recognition by an ABC transporter in Gram-positive bacteria.

molecular biology↗

bioRxiv2025

Biosynthesis, Structure, and Antibiotic Properties of Gelatinamin A, a Triculamin-like Lasso Peptide

Lasso peptides are structurally unique natural products endowed with high thermal and proteolytic stability, making them attractive as scaffolds for drug discovery. Recently, a new class of lasso peptides containing a second macrocycle, formed between a lysine sidechain and the C-terminus, was discovered, resulting in an even more compact architecture. Here, we report the first NMR structure of the class V lasso peptide, gelatinamin A. Using heterologous expression of the gelatinamin biosynthetic gene cluster (BGC) in Bacillus subtilis, we delineated the biosynthetic pathway through targeted gene deletions. We expressed and characterized the predicted transpeptidase, GelP, that catalyzes the formation of an isopeptide bond between Lys2 and the C-terminus and mediates the reversible conversion of gelatinamin B to gelatinamin A. In addition, we characterize GelT, an N-acetyltransferase that inactivates lasso peptide antimicrobial activity by acetylating a key lysine residue. Furthermore, we demonstrate that gelatinamin is highly potent against several important pathogens and that the activity is strongly bicarbonate-dependent. Finally, we propose a complete biosynthetic pathway for gelatinamin. The structural insight of gelatinamin A and the functional characterization of GelP provide the foundation for future discovery of class V lasso peptides and for engineering transpeptidases to modify other lasso peptide scaffolds.

biochemistry↗

bioRxiv2025

Functional and structural characterization of AtAbf43C: An exo-1,5-α-L-arabinofuranosidase from Acetivibrio thermocellus DSM1313

The Acetivibrio thermocellus DSM1313 genome codes for seven predicted glycoside hydrolase family 43 (GH43) enzymes, four of which remain uncharacterized. This study describes the function and structure of one such enzyme, AtAbf43C, from GH43 subfamily 26 (GH43_26) which acts as an -L-arabinofuranosidase (EC 3.2.1.55). AtAbf43C is active on para-nitrophenol--L-arabinofuranoside (pNPAra), with optimal activity observed at pH 5.5 and 65 . Multiple crystal structures of AtAbf43C were obtained, in which an N-terminal carbohydrate binding module family 42 (CBM42) domain displays a {beta}-trefoil type fold and the C-terminal GH43 domain displays a canonical 5-bladed {beta}-propeller motif. One structure, which was solved with two L-arabinofuranose molecules bound to {beta}- and {gamma}-subdomains of the CBM42, builds upon previous literature suggesting the -binding pocket of the AtAbf43C CBM42 is non-functional. Furthermore, structural alignment with the substrate bound structure of a closely homologous GH43_26 exo--1,5-arabinofuranosidase, SaAraf43A from Streptomyces avermitilis (PDB 3AKH), allowed for identification of the conserved catalytic triad via site-directed mutagenesis in AtAbf43C, as well as insight into the deep-narrow topology of the AtAbf43C binding pocket that suggested it would be active on similar arabino-oligosaccharide (AOS) substrates as SaAraf43A. Subsequent liquid chromatography-mass spectrometry (LC-MS) analysis of polysaccharides and oligosaccharides hydrolyzed by AtAbf43C provides experimental evidence confirming this enzyme acts in an exo manner primarily towards -1,5 linked arabino-oligosaccharides.

molecular biology↗

bioRxiv2024

Cysimiditides: RiPPs with a Zn-tetracysteine motif and aspartimidylation

Aspartimidylation is a post-translational modification found in multiple families of ribosomally synthesized and post-translationally modified peptides (RiPPs). We recently reported on the imiditides, a new RiPP family in which aspartimidylation is the class-defining modification. Imiditide biosynthetic gene clusters encode a precursor protein and a methyltransferase that methylates a specific Asp residue, converting it to aspartimide. A subset of imiditides harbor a tetracysteine motif, so we have named these molecules cysimiditides. Here, using genome mining we show that there are 56 putative cysimiditides predicted in publicly available genome sequences, all within actinomycetota. We successfully heterologously expressed two examples of cysimiditides and showed that the major products are aspartimidylated and that the tetracysteine motif is necessary for expression. Cysimiditides bind a Zn2+ ion, presumably at the tetracysteine motif. Using in vitro reconstitution of the aspartimidylation reaction, we show that Zn2+ is required for methylation and subsequent aspartimidylation of the precursor protein. An AlphaFold 3 model of the cysimiditide from Thermobifida cellulosilytica shows a hairpin structure anchored by the Zn2+-tetracysteine motif with the aspartimide site in the hairpin loop. An AlphaFold 3 model of this cysimiditide in complex with its cognate methyltransferase suggests that the methyltransferase recognizes the Zn2+-tetracysteine motif to correctly dock the precursor protein. Cysimiditides expand the set of experimentally-confirmed RiPPs harboring aspartimides, and represent the first RiPP class that has an obligate metal ion. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616296v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@2879corg.highwire.dtl.DTLVardef@152b63org.highwire.dtl.DTLVardef@1d3ad25org.highwire.dtl.DTLVardef@c8d63_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Discovery, Characterization, and Bioactivity of the Achromonodins: Lasso Peptides Encoded by Achromobacter

Through genome mining efforts, we discovered two lasso peptide biosynthetic gene clusters (BGCs) within two different species of Achromobacter, a genus that contains pathogenic organisms that can infect patients with cystic fibrosis. Using gene-refactored BGCs in E. coli, we heterologously expressed two lasso peptides, which we named achromonodin-1 and achromonodin-2. Achromonodin-1 is naturally encoded by certain isolates from the sputum of patients with cystic fibrosis. We solve the NMR structure of achromonodin-1, demonstrating that it is a threaded lasso peptide with a large loop and short tail structure, reminiscent of previously characterized lasso peptides that inhibit RNA polymerase (RNAP). We then show that achromonodin-1 inhibits RNAP in vitro and has potent but narrow-spectrum activity towards Achromobacter pulmonis, another isolate from the sputum of a cystic fibrosis patient. Our efforts expand the repertoire of antimicrobial lasso peptides and provide insights into how Achromobacter isolates from certain ecological niches may interact with each other.

biochemistry↗

bioRxiv2023

A large-scale bioinformatic study of graspimiditides and structural characterization of albusimiditide

Graspetides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that exhibits an impressive diversity in patterns of side chain-to-side chain {omega}-ester or {omega}-amide linkages. Recent studies have uncovered a significant portion of graspetides to contain an additional post-translational modification involving aspartimidylation catalyzed by an O-methyltransferase, predominantly found in the genomes of Actinomycetota. Here, we present a comprehensive bioinformatic analysis focused on graspetides harboring aspartimide for which we propose the name graspimiditides. From Protein BLAST results of 5,000 methyltransferase sequences, we identified 962 unique putative graspimiditides, which we further classified into eight main clusters based on sequence similarity along with several smaller clusters and singletons. The previously studied graspimiditides, fuscimiditide and amycolimiditide, are identified in this analysis; fuscimiditide is a singleton while amycolimiditide is in the fifth largest cluster. Cluster 1, by far the largest cluster, contains 641 members, encoded almost exclusively in the Streptomyces genus. To characterize an example of a graspimiditide in Cluster 1, we conducted experimental studies on the peptide from Streptomyces albus J1074, which we named albusimiditide. By tandem mass spectrometry, hydrazinolysis, and amino acid substitution experiments, we elucidated the structure of albusimiditide to be a large tetracyclic peptide with four {omega}-ester linkages generating a stem-loop structure with one aspartimide. The ester crosslinks form 22-, 46-, 22-, and 44-atom macrocycles, last of which, the loop, contains the enzymatically installed aspartimide. Further in vitro experiments revealed that the aspartimide hydrolyzes in a 3:1 ratio of isoaspartate to aspartate residues. Overall, this study offers a comprehensive insight into the diversity and structural features of graspimiditides, paving the way for future investigations of this unique class of natural product. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/545981v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@bd68f2org.highwire.dtl.DTLVardef@1350705org.highwire.dtl.DTLVardef@5ff593org.highwire.dtl.DTLVardef@1f3c4a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Genome Mining and Discovery of Imiditides, a Novel Family of RiPPs with a Class-defining Aspartimide Modification

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating class of natural products of ribosomal origins. In the past decade, various sophisticated machine learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Instead, we argue that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds for novel RiPP discovery. Leveraging that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized the C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, widely distributed in Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP associated PIMTs that recognize constrained peptides as substrates, the PIMT homolog in mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. The aspartimide moiety formed is unusually stable, leading to the accumulation of the aspartimidylated product in vivo. The substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experimental validations as well as an AlphaFold model prediction. Our study suggests that PIMT-mediated aspartimide formation is an underappreciated backbone modification strategy in RiPP biosynthesis, compared to the well-studied backbone rigidification chemistries, such as thiazol(in)e and oxazol(in)e formations. Additionally, our findings suggest that aspartimide formation in Gram-positive bacterial proteomes are not limited to spontaneous protein aging and degradation. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/536058v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@a36867org.highwire.dtl.DTLVardef@1b6044org.highwire.dtl.DTLVardef@14d3036org.highwire.dtl.DTLVardef@98666a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

A High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin

The Burkholderia cepacia complex (Bcc) is a group of bacteria including several opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. The natural product ubonodin is a lasso peptide with promising activity against several Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. In this study, we developed a high-throughput screen using next-generation sequencing to examine the fitness of a library of over 90,000 ubonodin variants, generating the most comprehensive dataset on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space, indicating certain residues that can tolerate amino acid substitutions and still retain activity. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a sub-micromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants identified in this study had a lower MIC against certain Bcc strains than many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.

biochemistry↗

bioRxiv2022

The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry

New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodins spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.

microbiology↗

bioRxiv2021

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/444711v1_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@158f0d2org.highwire.dtl.DTLVardef@2a6baeorg.highwire.dtl.DTLVardef@103a91dorg.highwire.dtl.DTLVardef@153dd99_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Fuscimiditide: a RiPP with {Omega}-Ester and Aspartimide Post-translational Modifications

Microviridins and other {omega}-ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/444834v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1c67f36org.highwire.dtl.DTLVardef@c9e774org.highwire.dtl.DTLVardef@17a7d62org.highwire.dtl.DTLVardef@bde61b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗