Hernández de la Torre, MarthaCovaleda Cortés, GiovanniMontesinos, LauraCovaleda, DanielaOrtiz, Juan C.Piñol, JaumeBautista Santa Cruz, José ManuelCastillo, J. PatricioReverter, DavidAvilés, Francesc Xavier2025-03-032025-03-032025Hernández de la Torre, M., Covaleda-Cortés, G., Montesinos, L., Covaleda, D., Ortiz, J. C., Piñol, J., Bautista, J. M., Castillo, J. P., Reverter, D., & Avilés, F. X. (2025). Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species. International Journal of Molecular Sciences, 26(3), 1150. https://doi.org/10.3390/ijms260311501661-659610.3390/ijms26031150https://hdl.handle.net/20.500.14352/118422Author Contributions: Conceptualization: M.H.d.l.T., G.C.-C., J.P.C., D.R. and F.X.A.; experimental acquisition, data analysis, writing, and editing: M.H.d.l.T., G.C.-C., L.M., D.C., J.C.O., J.P., J.M.B., J.P.C., D.R. and F.X.A.; general writing and revision: M.H.d.l.T., G.C.-C., J.P., J.M.B., D.R. and F.X.A.; corresponding author: F.X.A. All authors have read and agreed to the published version of the manuscriptProtease inhibitors are biomolecules with growing biotechnological and biomedical relevance, including those derived from plants. This study investigated strong trypsin inhibitors in quinoa, amaranth, and lupine seeds, plant grains traditionally used in Andean South America. Amaranth seeds displayed the highest trypsin inhibitory activity, despite having the lowest content of aqueous soluble and thermostable protein material. This activity, directly identified by enzymatic assay, HPLC, intensity-fading mass spectrometry (IF-MS), and MS/MS, was attributed to a single protein of 7889.1 Da, identified as identical in Amaranthus caudatus and A. hybridus, with a Ki of 1.2 nM for the canonical bovine trypsin. This form of the inhibitor, which is highly homogeneous and scalable, was selected, purified, and structurally–functionally characterized due to the high nutritional quality of amaranth seeds as well as its promising agriculture–biotech–biomed applicability. The protein was crystallized in complex with bovine trypsin, and its 3D crystal structure resolved at 2.85 Å, revealing a substrate-like transition state interaction. This verified its classification within the potato I inhibitor family. It also evidenced that the single disulfide bond of the inhibitor constrains its binding loop, which is a key feature. Cell culture assays showed that the inhibitor did not affect the growth of distinct plant microbial pathogen models, including diverse bacteria, fungi, and parasite models, such as Mycoplasma genitalium and Plasmodium falciparum. These findings disfavour the notion that the inhibitor plays an antimicrobial role, favouring its potential as an agricultural insect deterrent and prompting a redirection of its functional researchengAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article1422-0067https://doi.org/10.3390/ijms2603115039940919open access577.2Plant trypsin inhibitorsQuinoaAmaranthLupine seedsAmaranthus hybridusAmaranthus caudatusHPLCMS and X-ray analysisStructure–function characterisationPlant defenceBiología molecular (Biología)2415 Biología Molecular