Biology subjects

Mylne, J. S.

Publications and source records attributed to Mylne, J. S..

16 recordsLinked to original sources

bioRxiv2022

Structure-guided investigations into HMG-CoA reductase as a herbicide target

Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a new mode of action target for the development of herbicides. Here we present the crystal structure of a HMGR from Arabidopsis thaliana (AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enabled the rational design of specific HMGR inhibitors, for which we engineered a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.

plant biology↗

bioRxiv2021

Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds as well as heightened regulatory scrutiny. Of the known herbicide modes of action, 6-hydroxymethyl-7,8-dihydropterin synthase (DHPS) which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics - and while still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.6 [A] resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate asulam and the antibiotics sulfacetamide and sulfamethoxazole have herbicidal as well as antibacterial activity and explore the structural basis of their potency by modelling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and that pharmacokinetic or delivery differences between plants and microbes might be the best approaches to safeguard this mode of action.

biochemistry↗

bioRxiv2021

Inhibition of chloroplast translation as a new target for herbicides

The rise in herbicide resistance over recent decades threatens global agriculture and food security and so discovery of new modes of action is increasingly important. Here we reveal linezolid, an oxazolidinone antibiotic that inhibits microbial translation, is also herbicidal. To validate the herbicidal mode of action of linezolid we confirmed its micromolar inhibition is specific to chloroplast translation and did not affect photosynthesis directly. To assess the herbicide potential of linezolid, testing against a range of weed and crop species found it effective pre- and post-emergence. Using structure-activity analysis we identified the critical elements for herbicidal activity, but importantly also show, using antimicrobial susceptibility assays, that separation of antibacterial and herbicidal activities was possible. Overall these results validate chloroplast translation as a viable herbicidal target.

plant biology↗

bioRxiv2021

Solution NMR and racemic crystallography provide insights into a novel structural class of cyclic plant peptides

Head-to-tail cyclic and disulfide-rich peptides are natural products with applications in drug design. Among these are the PawS-Derived Peptides (PDPs) produced in seeds of the daisy plant family. PDP-23 is a unique member of this class in that it is twice the typical size and adopts two {beta}-hairpins separated by a hinge region. The {beta}-hairpins - both stabilised by a single disulfide bond - fold together into a V-shaped tertiary structure creating a hydrophobic core. In water two PDP-23 molecules merge their hydrophobic cores to form a square prism quaternary structure. Here, we synthesised PDP-23 and its enantiomer comprising all D-amino acids, which allowed us to confirm these solution NMR structural data by racemic crystallography. Furthermore, we discovered the related PDP-24. NMR analysis showed that PDP-24 does not form a dimeric structure and it has poor water solubility, but in less polar solvents adopts near identical secondary and tertiary structure to PDP-23. The natural role of these peptides in plants remains enigmatic, as we did not observe any antimicrobial or insecticidal activity. However, the plasticity of these larger PDPs and their ability to change structure under different conditions make them appealing peptide drug scaffolds.

biochemistry↗

bioRxiv2021

Structural Characterization of the PawL-Derived Peptide Family, an Ancient Subfamily of Orbitides

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic but non-disulfide bonded plant peptides, known as orbitides, has been relatively ignored. A recently discovered subfamily of orbitides is the PawL-Derived Peptides (PLPs), produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the familys structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive and systematic characterization of the PLP family. Nine PLPs were chosen and prepared by solid phase peptide synthesis. Their structural features were studied using solution NMR spectroscopy and seven were found to possess regions of backbone order. Ordered regions consist of {beta}-turns, with some PLPs adopting two well-defined {beta}-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse molecular scaffolds. None of these nine PLPs showed antibacterial or antifungal activity.

biochemistry↗

bioRxiv2021

Herbicidal activity of fluoroquinolone derivatives

Development of herbicides with novel modes of action are crucial for weed control and to hinder herbicide resistance. An attractive novel herbicidal target is plant DNA gyrase, which has been demonstrated to be effectively inhibited by the known antimicrobial ciprofloxacin. Despite this good herbicidal activity ciprofloxacin is not suitable as a herbicide due to its antimicrobial activity, therefore, a diverse library of analogues was analysed to gain insight into the aspects required for herbicidal activity. This analysis revealed significant structural modifications were tolerated and that the fluoride at C-6 and a cyclic amino group at C-7 were not crucial for herbicidal activity. The analysis also revealed that these modifications also affected the antibacterial activity with one compound demonstrating good herbicidal activity and weak antibacterial activity, against both Gram-positive and Gram-negative bacteria.

plant biology↗

bioRxiv2021

A systematic approach for finding herbicide synergies

Combining herbicides into a double dose is a common approach to overcome the potential for herbicide resistance by weeds. Many herbicide mixtures can be antagonistic and they are rarely synergistic. Here, 24 commercial herbicides, each representing a mode of action were used to create a matrix of all 276 unique combinations to search for new synergies in agar with Arabidopsis thaliana. Herbicides were used at an appropriate sub-lethal dose such that any synergies gave visible growth inhibition. We found five synergies including three new ones, namely mesotrione-norflurazon, mesotrione-clethodim and paraquat-clomazone. All three new synergies were reproducible in soil-grown conditions. Interestingly, all three new combinations included a bleaching herbicide, suggesting synergy might be a class specific phenomenon. We also found that mesotrione-norflurazon and mesotrione-clethodim combinations remained synergistic against lettuce (Lactuca sativa), but not tef (Eragrostis tef). Our study shows that screening herbicide mixtures against A. thaliana is an efficient approach for finding rare herbicide synergies.

plant biology↗

bioRxiv2020

A Chameleonic Macrocyclic Peptide with Drug Delivery Applications

Head-to-tail cyclized peptides are intriguing natural products with unique properties. The PawS-Derived Peptides (PDPs) are produced from precursors of seed storage albumins in species of the daisy family. Here we report an unusually large PDP with two disulfide bonds, identified from seeds of Zinnia elegans. In water, synthetic PDP-23 forms a unique dimeric structure in which two monomers containing two {beta}-hairpins cross-clasp and enclose a hydrophobic core, creating a square prism. This stable dimer can be split and each monomer unfolds to a V-shape in micelles or organic solvents. This chameleonic character is unusual for disulfide-rich peptides and engenders PDP-23 with potential for cell delivery and accessing novel targets. We demonstrated this by conjugating a rhodamine dye to the PDP-23 scaffold, creating a stable, cell-penetrating inhibitor of the P-glycoprotein drug efflux pump.

biochemistry↗

bioRxiv2020

Designing safe and potent herbicides with the cropCSM online resource

Herbicides have revolutionised weed management, increased crop yields and improved profitability allowing for an increase in worldwide food security. Their widespread use, however, has also led to not only a rise in resistance but also concerns about their environmental impact. To help identify new, potent, non-toxic and environmentally safe herbicides we have employed interpretable predictive models to develop the online tool cropCSM (http://biosig.unimelb.edu.au/crop_csm).

bioinformatics↗

bioRxiv2020

Structural basis for a natural circular permutation in proteins

Over 30 years ago, an intriguing post-translational modification was discovered to be responsible for creating concanavalin A (conA), a carbohydrate-binding protein found in the seeds of jack bean (Canavalia ensiformis) and commercially used for carbohydrate chromatography. Biosynthesis of conA involves what was then an unprecedented rearrangement in amino acid sequence, whereby the N-terminal half of the gene-encoded conA precursor is swapped to become the C-terminal half of conA. The cysteine protease, asparaginyl endopeptidase (AEP), was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of conA circular permutation, we generated a recombinant jack bean conA precursor (pro-conA) plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 [A] and 2.7 [A] respectively. By reconstituting the biosynthesis of conA in vitro, we prove CeAEP1 alone can perform both the cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both these reactions. Biophysical assays illustrated that conA is more thermally and pH stable than pro-conA, consistent with fewer intermolecular interactions between subunits in the pro-conA crystal structure. These findings elucidate the consequences of circular permutation in the only post-translation example known to occur in nature.

biochemistry↗

bioRxiv2020

Refining physico-chemical rules for herbicides using an antimalarial library

Successful herbicides, like drugs, have physico-chemical properties that usually fall within certain limits. A recent analysis of 334 herbicides showed similar properties to the rule of five for human orally-delivered drugs, but herbicides diverged from this for proton donors, partition coefficients and molecular weight. To refine rules for herbicides, we exploited the close evolutionary relationship between P. falciparum and plants by screening the Malaria Box, a 400-compound library composed of novel chemical scaffolds with activity against blood stage malaria parasite Plasmodium falciparum. A high proportion (52 of 400) were herbicidal to Arabidopsis thaliana on agar plates. Thirty-nine of these 52 herbicidal compounds were tested on soil and 16 compounds were herbicidal. These data were used to predict whether a herbicidal hit found on agar will work on soil-grown plants. The physico-chemical parameters were weighted to logP and formal charge and used to generate weighted scores to a large chemical library of liver-stage effective antimalarial leads. Of the six top-scoring compounds, one had a potency comparable to commercial herbicides. This novel compound MMV1206386 had no close structural matches among commercial herbicides. Physiological profiling suggested that MMV1206386 has a new mode of action and overall demonstrates how weighted rules can help during herbicide discovery programs.

plant biology↗

bioRxiv2020

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides

Cyclic peptides are reported to have antibacterial, antifungal and other bioactivities. Several genera of the Rutaceae family are known to produce orbitides, which are small head-to-tail cyclic peptides composed of proteinogenic amino acids and lacking disulfide bonds. Melicope xanthoxyloides is an Australian rain forest tree of the Rutaceae family in which evolidine - the first plant cyclic peptide - was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, but here we use tandem mass spectrometry to rediscover evolidine and using de novo transcriptomics we show its biosynthetic origin to be from a short precursor just 48 residues in length. In all, seven M. xanthoxyloides orbitides were found and they had atypically diverse C-termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides. Two of the novel orbitides were studied by nuclear magnetic resonance spectroscopy and although one had definable structure, the other did not. By mining RNA-seq and whole genome sequencing data from other species, it was apparent that a large and diverse family of peptides is encoded by sequences like these across the Rutaceae.

biochemistry↗

bioRxiv2020

Defining the familial fold of the vicilin-buried peptide family

Plants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We here present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to adopt a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladder-like connectivity of their disulfide bonds. Inter-helix interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the anti-parallel -helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function, however their diverse structural features despite a common fold suggest additional bioactivities yet to be revealed are likely.

biochemistry↗

bioRxiv2019

Novel cyclic peptides in seeds of Annona muricata are ribosomally synthesised

Small, cyclic peptides are reported to have many bioactivities. In bacteria and fungi they can be made by non-ribosomal peptide synthetases, but in plants they are exclusively ribosomal. Cyclic peptides from the Annona genus possess cytotoxic and anti-inflammatory activities, but their biosynthesis is unknown. The medicinal soursop plant, Annona muricata, contains annomuricatins A (cyclo-PGFVSA) and B (cyclo-PNAWLGT). Here, using de novo transcriptomics and tandem mass spectrometry, we identify a suite of short transcripts for precursor proteins for ten validated annomuricatins, nine of which are novel. In their precursors, annomuricatins are preceded by an absolutely conserved Glu and each peptide sequence has a conserved proto-C-terminal Pro, revealing parallels with the segetalin orbitides from the seed of Vaccaria hispanica, which are processed through ligation by a prolyl oligopeptidase in a transpeptidation reaction.

biochemistry↗

bioRxiv2019

De Novo Peptide Sequencing Reveals a Vast Cyclopeptidome in Human Gut and Other Environments

Cyclic and branch cyclic peptides (cyclopeptides) represent an important class of bioactive natural products that include many antibiotics and anti-tumor compounds. However, little is known about cyclopeptides in the human gut, despite the fact that humans are constantly exposed to them. To address this bottleneck, we developed the CycloNovo algorithm for de novo cyclopeptide sequencing that employs de Bruijn graphs, the workhorse of DNA sequencing algorithms. CycloNovo reconstructed many new cyclopeptides that we validated with transcriptome, metagenome, and genome mining analyses. Our benchmarking revealed a vast hidden cyclopeptidome in the human gut and other environments and suggested that CycloNovo offers a much-needed step-change for cyclopeptide discovery. Furthermore, CycloNovo revealed a wealth of anti-microbial cyclopeptides from food that survive the complete human gastrointestinal tract, raising the question of how these cyclopeptides might affect the human microbiome.\n\nSIGNIFICANCEThe golden age of antibiotics was followed by a decline in the pace of antibiotics discovery in the 1990s. The key prerequisite for the resurgence of antibiotics research is the development of a computational discovery pipeline for antibiotics sequencing. We describe such pipeline for cyclic and branch cyclic peptides (cyclopeptides) that represent an important class of bioactive natural products such as antibiotics and anti-tumor compounds. Our CycloNovo algorithm for cyclopeptide sequencing reconstructed many new cyclopeptides that we validated with transcriptome, metagenome, and genome mining analyses. CycloNovo revealed a wealth of anti-microbial cyclopeptides from food that survive the complete human gastrointestinal tract, raising the question of how these cyclopeptides might affect the human microbiome.

bioinformatics↗

bioRxiv2018

The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs have attracted attention for their macrocyclization reactions and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

biophysics↗