Biology subjects

Buchko, G. W.

Publications and source records attributed to Buchko, G. W..

4 recordsLinked to original sources

bioRxiv2026

A truncated soil phage catechol 1,2-dioxygenase illustrates how viruses preserve and disseminate auxiliary catalytic functions in the soil microbiome

Bacteriophages can rewire host chemistry via auxiliary viral genes (AVGs). Using metagenomic and metatranscriptomic data from the native soil microbiome, we identified transcriptionally active AVGs, including a viral catechol 1,2-dioxygenase (V-C12DO). V-C12DO shares [~]40% sequence identity with its nearest bacterial homologs and lacks the helical dimerization domain. Despite truncation, V-C12DO retains more than [~]25% of the global consensus residues compared to C12DOs across domains of life, including the two tyrosines and two histidines that coordinate the non-heme Fe(III) active site. A 1.7 [A] crystal structure also showed the conservation of the canonical {beta}-sandwich scaffold for the iron. We next confirmed that V-C12DO cleaves catechol and remains highly active across a broad range of temperatures (30-60 {degrees}C), pH (5.5-9), and salinity (up to 2 M), exceeding those of known bacterial CD12Os. This work shows that truncated phage enzymes preserve the core catalytic chemistry and potentially further expand host metabolic versatility across dynamic environmental conditions.

microbiology↗

bioRxiv2024

Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted-repeat lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form 2 or 3 intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +9 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides structural framework for design of novel, potent antifungal peptides for agricultural use. Author SummaryFungal pathogens cause significant pre-harvest and post-harvest losses of crop yield, making them a serious biological threat to global food security. Chemical fungicides are effective in controlling fungal diseases across various crops. However, rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has created an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Nodule-specific cysteine-rich (NCR) peptides expressed in the inverted-repeat lacking clade legume plants exhibit potent antifungal activity; however, their modes of action (MoA) are poorly understood. Particularly, the specific contribution of disulfide pairing to the potency and spectrum of antifungal activity against fungal plant pathogens and MoA of these peptides remains to be identified. Chickpea NCR13 expressed in P. pastoris generates two peptide variants that differ in their disulfide cross-linking pattern. These variants exhibit striking differences in their three-dimensional structures and potency of antifungal activity against multiple fungal pathogens and MoA. They also differ in their ability to confer resistance to gray mold in pepper and tomato plants. Our study highlights the major impact a specific pattern of disulfide pairing can have on the in vitro and in planta antifungal activity of an NCR peptide.

plant biology↗

bioRxiv2021

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 M range in vitro. Furthermore, Exebryl-1, a {beta}-amyloid anti-aggregation molecule for Alzheimers therapy, was shown to have antiviral activity between 10 to 66 M, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral. Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

microbiology↗

bioRxiv2020

Antifungal symbiotic peptide NCR044.1 exhibits unique structure and multi-faceted mechanisms of action that confer plant protection

NCR044.1 is a 36-amino acid nodule-specific cysteine-rich antimicrobial peptide expressed in the developing nodules of Medicago truncatula. Here, we determined its unique NMR structure to be largely disordered, one four-residue -helix and one three-residue anti-parallel {beta}-sheet stabilized by two disulfide bonds, suggesting it is highly dynamic. NCR044.1 exhibited potent fungicidal activity against multiple plant fungal pathogens. It breached the fungal plasma membrane, bound to multiple phosphoinositides, and induced reactive oxygen species. Time-lapse confocal and super-resolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, and subsequent accumulation in the cytoplasm with elevated levels in nucleoli. Nucleolar localization of NCR044.1 was unique amongst plant antifungal peptides, suggesting its potential interaction with ribosomes and inhibition of translation. Spray-applied NCR044.1 significantly reduced gray mold disease symptoms caused by the fungal pathogen Botrytis cinerea in tomato plants and post-harvest products demonstrating its potential as a spray-on peptide-based biofungicide.

plant biology↗