Biology subjects

Seefeldt, L.

Publications and source records attributed to Seefeldt, L..

5 recordsLinked to original sources

bioRxiv2026

A high-throughput compound screen identifies multiple druggable targets in Plasmodium falciparum transmission stages

Most antimalarials are ineffective against the sexual transmission stages, known as gametocytes, of the malaria parasite Plasmodium falciparum. Their low sensitivity to drugs is attributed to limited compound uptake and a poorly understood form of cellular quiescence. Our current understanding of druggable transmission-blocking processes is therefore limited. Based on genetically engineered parasites that facilitate the mass production of synchronous mature gametocytes, we developed a high throughput drug screening platform that allowed us to test more than 50,000 compounds for gametocytocidal effects in one day. By screening a diversity-oriented library, we identified over 40 molecules that kill mature gametocytes in the low nanomolar range. Using resistance selection coupled to whole genome sequencing and drug-target interaction modelling, we followed up on three chemically tractable compounds that are also highly active against asexual parasites and prevent gametocyte transmission to mosquitoes. We show that the compound ONX-0914, a specific inhibitor of the {beta}5i/LMP7 subunit of human immunoproteasomes, targets the parasite proteasomal {beta}5 subunit. In contrast, the compounds CR-1-31-B and brusatol interfere with translation by targeting eukaryotic initiation factor 4A (eIF4A) and the peptidyl transferase center (PTC) of the 80S ribosome, respectively. Interestingly, parasite resistance to brusatol, a broad-spectrum antitumor drug, is linked to the differential modification of specific rRNA bases near the ribosomal A-site, mediated by altered base specificity of a rRNA methyltransferase. In summary, we successfully combined high-throughput compound screening with drug target deconvolution to reveal the targets and mode-of-action for three potent gametocytocidal molecules and discover the mechanism of resistance to the anti-tumorigenic drug brusatol. In addition to critically advancing our understanding of mature gametocyte biology and druggable processes in P. falciparum transmission stages, our observations made with brusatol-resistant parasites may become relevant for anti-cancer drug research.

molecular biology↗

bioRxiv2025

Persistent nitrogenase isotope signatures over two billion years of engineered evolution

Nitrogen isotope fractionation ({varepsilon}15N) in sedimentary rocks has provided evidence for biological nitrogen fixation, and thus primary productivity, on the early Earth. However, the extent to which molecular evolution has influenced the isotopic signatures of nitrogenase, the enzyme that catalyzes the conversion of atmospheric nitrogen (N2) to bioavailable ammonia, remains unresolved. Here, we reconstruct and experimentally characterize a library of synthetic ancestral nitrogenase genes, spanning over 2 billion years of evolutionary history. By engineering modern microbes to express these ancient nitrogenases, we assess the resulting {varepsilon}15N values under controlled laboratory conditions. All engineered strains exhibit {varepsilon}15N values within a narrow range comparable to that of modern microbes, suggesting that molybdenum (Mo)-dependent nitrogenase has been largely invariant throughout evolutionary time since the origins of this pathway. These results confirm the robustness of N-isotope biosignatures in the ancient rock record and bolster their utility in the search for life in extraterrestrial environments.

evolutionary biology↗

bioRxiv2024

Nitrogenase structural evolution across Earth s history

Life on Earth is about 4 billion years old--nearly as old as the planet itself. Over this immense timespan, living systems and their biomolecules have both adapted to and driven profound changes in the Earths environment. Among these, certain critical enzymes emerged early and have persisted through planetary extremes. Here, we implement an integrated approach to investigate the structural evolution of nitrogenase, an ancient and globally essential enzyme responsible for biological nitrogen fixation. Despite the ecological diversity of its host microbes, nitrogenase retains strict functional constraints, including extreme oxygen sensitivity, high energy demands, and substrate availability. We combined phylogenetics, ancestral sequence reconstruction, protein crystallography and deep-learning based structural prediction to resurrect nearly three billion years of nitrogenase structural history. This effort represents the first effort to predict the full set of extant and ancestral structures along the evolutionary tree of a single enzyme, yielding over 5000 structural models. Our framework lays a foundation for reconstructing key structural constraints that shape protein evolution and examining ancient enzymes within the broader context of phylogenetic relationships and environmental transitions across geological timescales.

evolutionary biology↗

bioRxiv2024

The Origins of ATP Dependence in Biological Nitrogen Fixation

Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg2+, can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the key N2-reducing enzyme nitrogenase. We show that the ancestor has a strict requirement for ATP and its hydrolysis is coupled to electron transfer for N2 reduction. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme, and underscore how biomolecular constraints can entirely decouple cofactor selection from environmental availability.

evolutionary biology↗

bioRxiv2022

Conservation of nitrogenase functionality over long timescales

The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, that is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism. IMPACT STATEMENTThe enzymatic mechanism for dinitrogen reduction is an ancient feature of nitrogenases that persisted over hundreds of millions of years.

evolutionary biology↗