Biology subjects

Fisher, M. F.

Publications and source records attributed to Fisher, M. F..

9 recordsLinked to original sources

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

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↗

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

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↗