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Pluskal, T.

Publications and source records attributed to Pluskal, T..

11 recordsLinked to original sources

BoltzGen: Toward Universal Binder Design

We introduce BoltzGen, an all-atom generative model for designing proteins and peptides across all modalities to bind a wide range of biomolecular targets. BoltzGen builds strong structural reasoning capabilities about target-binder interactions into its generative design process. This is achieved by unifying design and structure prediction, resulting in a single model that also reaches state-of-the-art folding performance. BoltzGens generation process can be controlled with a flexible design specification language over covalent bonds, structure constraints, binding sites, and more. We experimentally validate these capabilities in eight diverse design campaigns with functional and affinity readouts across 26 targets. In our experiments, binder modalities span from nanobodies to disulfide-bonded peptides, and targets from disordered proteins to small molecules. In particular, we identify nanobody binders for novel targets with low similarity to proteins with already known bound structures. We release model weights, data, and both inference and training code at: https://github.com/HannesStark/boltzgen.

bioengineering↗

A miniaturized, high-throughput buffer-centric method for protein solubility screening

Efficient access to soluble recombinant proteins remains a major bottleneck in biochemical and structural studies. We describe an aqueous, solvent-centric, fully miniaturized 96-well workflow to screen extraction conditions that preserve soluble recombinant protein during lysis and clarification in a single working day. Liquid-nitrogen-frozen E. coli pellets are cryogenically bead-milled with stainless-steel beads, retaining the native intracellular milieu while ensuring uniform disruption. The resulting wet, frozen cell powder can therefore be extracted with user-defined solvent, enabling systematic exploration of pH, ionic strength, detergents, and chaotropes. Protein solubility is assessed by a 1 {micro}L chromogenic anti-His dot-blot. We demonstrate the use of the protocol by solubilizing a set of highly challenging de novo-generated proteins and show that dot-blot intensity provides a practical semi-quantitative proxy for successful extraction of soluble proteins. We also provide experimentally supported guidelines on the influence of solvent reagents on subsequent steps of protein production, SDS-PAGE, and Ni-NTA purification. This workflow is compatible with upstream genetic solubility-enhancement, chassis- and cultivation-based strategies and enables direct transition from screening hits to scale-up. Because the workflow uses standard molecular biology equipment and inexpensive consumables, it can be readily adopted or automated in most laboratories.

biochemistry↗

Entry Steps in the Biosynthetic Pathway to DiterpenoidAlkaloids in Delphinium grandiflorum and Aconitum plicatum

Both the terpenoid and alkaloid classes of specialized metabolites have received considerable attention for their wide range of practical applications, however, few examples have been identified of these classes intersecting. The diterpenoid alkaloids are one such example of nitrogen-containing terpenoids which are found throughout many independent plant lineages, but primarily within the Aconitum (Wolfs-Bane) and Delphinium (Larkspur) genera. While there is considerable interest in these compounds for their wide range of bioactivities, their structural complexity often precludes their production through chemical synthesis, and little progress has been made towards elucidation of their biosynthetic pathways. Here, we employ a comparative transcriptomics approach to identify six enzymatic steps in the biosynthesis of atisinium, conserved across both Delphinium grandiflorum and Aconitum plicatum. Key to this pathway is a reductase which selectively incorporates ethanolamine over ethylamine into the diterpenoid scaffold. While the majority of diterpenoid alkaloids contain an ethylamine moiety, we demonstrate through isotope labeling in Aconitum callus cultures and a computational metabolomics approach that ethanolamine is, unintuitively, the preferred source of nitrogen for these metabolites. Identification of these enzymes and production of a key intermediate in a heterologous host paves the way for biosynthetic production of this group of metabolites with promise for medicinal applications.

biochemistry↗

MARTS-DB: A Database of Mechanisms And Reactions of Terpene Synthases

BackgroundTerpene synthases (TPSs) are enzymes that catalyze some of the most complex reactions in nature - the cyclizations of terpenes, which form the carbon backbones to the largest group of natural products, the terpenoids. On average, more than half of the carbon atoms in a terpene scaffold undergo a change in connectivity or configuration during these enzymatic cascades. Understanding TPS reaction mechanisms remains challenging, often requiring intricate computational modeling and isotopic labelling studies. Moreover, the relationship between TPS sequence and catalytic function is difficult to decipher, while data-driven approaches remain limited due to a lack of comprehensive, high-quality data sources. MainWe introduce the Mechanisms And Reactions of Terpene Synthases DataBase (MARTS-DB) - a manually curated, structured, and searchable database that integrates TPS enzymes, the terpenes they produce, and their detailed reaction mechanisms. MARTS-DB includes over 2,600 reactions catalyzed by 1,334 annotated enzymes from across all domains of life, with reaction mechanisms mapped as stepwise cascades for more than 400 terpenes. Accessible at https://www.marts-db.org, the database provides advanced search functionality and supports full dataset downloads in machine-readable formats. It also encourages community contributions to promote continuous growth. ConclusionUser-friendly and comprehensive, MARTS-DB enables the systematic exploration of TPS catalysis, opening new avenues for computational analysis and machine learning, as recently demonstrated in the prediction of novel TPSs.

bioinformatics↗

Remarkable diversity of alkaloid scaffolds in Piper fimbriulatum

Plant specialized metabolites play key roles in diverse physiological processes and ecological interactions. Identifying structurally novel metabolites, as well as discovering known compounds in new species, is often crucial for answering broader biological questions. The Piper genus (Piperaceae family) is known for its special phytochemistry and has been extensively studied over the past decades. Here, we investigated the alkaloid diversity of Piper fimbriulatum, a myrmecophytic plant native to Central America, using a metabolomics workflow that combines untargeted LC-MS/MS analysis with a range of recently-developed computational tools. Specifically, we leverage open MS/MS spectral libraries and metabolomics data repositories for metabolite annotation, guiding isolation efforts towards structurally-new compounds (i.e., dereplication). As a result, we identified several alkaloids belonging to 5 different classes and isolated one novel seco-benzylisoquinoline alkaloid featuring a linear quaternary amine moiety that we named fimbriulatumine. Notably, many of the identified compounds were never reported in Piperaceae plants. Our findings expand the known alkaloid diversity of this family, and demonstrate the value of revisiting well-studied plant families using state-of-the-art computational metabolomics workflows to uncover previously overlooked chemodiversity. To contextualize our findings into a broader biological context, we employed a workflow for automated mining of literature reports of the identified alkaloid scaffolds and mapped the results onto the angiosperm tree of life. By doing so, we highlight the remarkable alkaloid diversity within the Piper genus and provide a framework for generating hypotheses on the biosynthetic evolution of these specialized metabolites. Many of the computational tools and data resources used in this study remain underutilized within the plant science community. This manuscript demonstrates their potential through a practical application and aims to promote broader accessibility to untargeted metabolomics approaches. Significance StatementWe combine untargeted metabolomics with a range of recently developed computational tools to uncover a previously overlooked diversity of alkaloid scaffolds in Piper fimbriulatum. Our findings demonstrate the potential of revisiting well-studied plant families using state-of-the-art computational metabolomics workflows to uncover previously overlooked chemodiversity.

plant biology↗

Discovery and isolation of novel capsaicinoids and their TRPV1-related activity

Chilis contain capsaicin and other structurally related molecules known as capsaicinoids. Capsaicins target protein, the transient receptor potential cation channel subfamily V member 1 (TRPV1), has been linked to many post-activation effects, including changes in metabolism and pain sensation. Capsaicinoids also bind to TRPV1, but current studies often disregard non-capsaicin interactions. To fill in these gaps, we screened 40 different chili varieties derived from four Capsicum species by means of untargeted metabolomics and a rat TRPV1 (rTRPV1) calcium influx activation assay. The resulting capsaicinoid profiles were specific to each variety but only partially corresponded with species delimitations. Based on rTRPV1 activation elicited by crude chili extracts, capsaicinoids act in an additive manner and a capsaicinoid profile can serve as a gauge of this activation. In addition, we isolated eighteen capsaicinoids, including five previously unreported ones, and confirmed their structure by NMR and MS/MS. We then tested rTRPV1 activation by 23 capsaicinoids and three related compounds. This testing revealed that even slight deviations from the structure of capsaicin reduce the ability to activate the target, with a mere single hydroxylation on the acyl tail reducing potency towards rTRPV1 by more than 100-fold. In addition, we tested how rTRPV1 activity changes in the presence of capsaicin together with non-activating capsaicin analogs and weakly activating capsaicinoids and found both classes of molecules to positively modulate the effects of capsaicin. This demonstrates that even such compounds have measurable pharmacological effects, making a case for the use and study of natural chili extracts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/620944v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d56d02org.highwire.dtl.DTLVardef@e7a66dorg.highwire.dtl.DTLVardef@5f409forg.highwire.dtl.DTLVardef@18b88f7_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Smith, J. (2025) https://BioRender.com/a61h668 HighlightsO_LI5 novel capsaicinoids (vanilloids) and 13 other capsaicinoids were isolated from chilis. C_LIO_LIThe slightest deviation in the capsaicin structure results in decreased TRPV1 activity. C_LIO_LIA single hydroxylation can reduce potency to TRPV1 100-fold. C_LIO_LINon-pungent vanilloids modulate capsaicin-TRPV1 activity. C_LI

pharmacology and toxicology↗

Discovery and Characterization of Terpene Synthases Powered by Machine Learning

The exponential growth of uncharacterized enzyme sequences in genomic repositories demands novel tools for functional annotation. Here, we combined alignment-driven structural domain analysis with protein language models to create EnzymeExplorer, a machine-learning pipeline for enzyme function prediction. We applied this approach to terpene synthases (TPSs), which present an ideal model case because they catalyze complex carbocationic rearrangements with unpredictable product outcomes. We detected new structural domains and achieved significantly higher average precision than existing methods for function prediction. By analyzing the UniRef90 database, we identified TPSs overlooked by existing computational methods. Remarkably, we discovered and experimentally confirmed three archaeal TPSs, expanding the known taxonomic distribution of TPS catalysis to a new domain of life. Further in silico screening of archaeal proteomes revealed that terpene biosynthesis is widespread across Archaea. Our approach offers a powerful framework for characterizing enzyme "dark matter" in the rapidly expanding genomic and metagenomic datasets.

biochemistry↗

POMBOX: a fission yeast toolkit for molecular and synthetic biology

Schizosaccharomyces pombe is a popular model organism in molecular biology and cell physiology. With its ease of genetic manipulation and growth, supported by in-depth functional annotation in the PomBase database and genome-wide metabolic models, S. pombe is an attractive option for synthetic biology applications. However, S. pombe currently lacks modular tools for generating genetic circuits with more than one transcriptional unit. We have developed a toolkit to address this issue. Adapted from the MoClo- YTK plasmid kit for Saccharomyces cerevisiae and using the same Golden Gate grammar, our POMBOX toolkit is designed to facilitate the fast, efficient and modular construction of genetic circuits in S. pombe. It allows for interoperability when working with DNA sequences that are functional in both S. cerevisiae and S. pombe (e.g. protein tag, antibiotic resistance cassette, coding sequences). Moreover, POMBOX enables the modular assembly of multi-gene pathways and increases possible pathway length from 6 to 12 transcriptional units. We also adapted the stable integration vector homology arms to Golden Gate assembly and tested the genomic integration success rate depending on different sequence sizes, from four to twenty-four kilobases. We included fourteen S. pombe promoters that we characterized for two fluorescent proteins, in both minimal defined media (EMM2) and complex media (YES). Then we tested six S. cerevisiae and six synthetic terminators in S. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes in S. pombe to produce specialized metabolite precursors, namely methylxanthine, amorpha-4,11-diene and cinnamic acid from the purine, mevalonate and amino acid pathways.

synthetic biology↗

Ion Identity Molecular Networking in the GNPS Environment

Molecular networking connects tandem mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. To connect ion species of the same molecule, we developed Ion Identity Molecular Networking. These new relationships improve network connectivity, are shown to reveal novel ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral libraries.

bioinformatics↗

PBS3 and EPS1 complete salicylic acid biosynthesis from isochorismate in Arabidopsis

Salicylic acid (SA) is an important phytohormone mediating both local and systemic defense responses in plants. Despite over half a century of research, how plants biosynthesize SA remains unresolved. In Arabidopsis, a major part of SA is derived from isochorismate, a key intermediate produced by the isochorismate synthase (ICS), which is reminiscent of SA biosynthesis in bacteria. Whereas bacteria employ an isochorismate pyruvate lyase (IPL) that catalyzes the turnover of isochorismate to pyruvate and SA, plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism. Combining genetic and biochemical approaches, we delineated the SA biosynthetic pathway downstream of isochorismate in Arabidopsis. We show that PBS3, a GH3 acyl adenylase-family enzyme important for SA accumulation, catalyzes ATP- and Mg2+-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate isochorismoyl-glutamate A. Moreover, EPS1, a BAHD acyltransferase-family protein with previously implicated role in SA accumulation upon pathogen attack, harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase (IPGL) activity that produces SA from the isochorismoyl-glutamate A substrate. Together, PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis, which is distinct from how SA is biosynthesized in bacteria. This study closes a major knowledge gap in plant SA metabolism and would help develop new strategies for engineering disease resistance in crop plants.

plant biology↗

The biosynthetic origin of psychoactive kavalactones in kava

For millennia, humans have used plants for medicinal purposes. However, our limited understanding of plant biochemistry hinders the translation of such ancient wisdom into modern pharmaceuticals1. Kava (Piper methysticum) is a medicinal plant native to the Polynesian islands with anxiolytic and analgesic properties supported by over 3,000 years of traditional use as well as numerous recent clinical trials2-5. The main psychoactive principles of kava, kavalactones, are a unique class of polyketide natural products known to interact with central nervous system through mechanisms distinct from those of the prescription psychiatric drugs benzodiazepines and opioids6,7. Here we report de novo elucidation of the biosynthetic pathway of kavalactones, consisting of seven specialized metabolic enzymes. Based on phylogenetic and crystallographic analyses, we highlight the emergence of two paralogous styrylpyrone synthases, both of which have neofunctionalized from an ancestral chalcone synthase to catalyze the formation of the kavalactone scaffold. Structurally diverse kavalactones are then biosynthesized by subsequent regio- and stereo-specific tailoring enzymes. We demonstrate the feasibility of engineering heterologous production of kavalactones and their derivatives in bacterial, yeast, and plant hosts, thus opening an avenue towards the development of new psychiatric therapeutics for anxiety disorders, which affect over 260 million people globally8.

plant biology↗