biostudies-literatureLiao JDeutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft (German Research Foundation)King Abdullah University of Science and Technology3065https://www.ebi.ac.uk/biostudies/studies/S-EPMC11954892biostudies-literatureUnknown16(1)Enzymes are essential catalysts in biological systems. Substrate inhibition, once dismissed, is now observed in 20% of enzymes¹ and is attributed to the formation of an unproductive enzyme-substrate complex, with no structural evidence of unproductivity provided to date^1-6. This study uncovers the molecular mechanism of substrate inhibition in tobacco glucosyltransferase NbUGT72AY1, which transfers glucose to phenols for plant protection. The peculiarity that β-carotene strongly attenuates the substrate inhibition of NbUGT72AY1, despite being a competitive inhibitor, allows to determine the conformational changes that occur during substrate binding in both active and substrate-inhibited complexes. Crystallography reveals structurally different ternary enzyme-substrateNature communicationsβ-Carotene alleviates substrate inhibition caused by asymmetric cooperativity.PMC11954892SCHW 634/34-1Steinchen WHoffmann TDKurze ESong CDi Pizio ANicoli ACatici DAMKuttler CShahul Hameed UFLiao JSchwab WGAssaad-Gerbert FArold STSun GHoffmann Tfalseβ-Carotene alleviates substrate inhibition caused by asymmetric cooperativity.Enzymes are essential catalysts in biological systems. Substrate inhibition, once dismissed, is now observed in 20% of enzymes¹ and is attributed to the formation of an unproductive enzyme-substrate complex, with no structural evidence of unproductivity provided to date^1-6. This study uncovers the molecular mechanism of substrate inhibition in tobacco glucosyltransferase NbUGT72AY1, which transfers glucose to phenols for plant protection. The peculiarity that β-carotene strongly attenuates the substrate inhibition of NbUGT72AY1, despite being a competitive inhibitor, allows to determine the conformational changes that occur during substrate binding in both active and substrate-inhibited complexes. Crystallography reveals structurally different ternary enzyme-substrate2025-01-01T00:00:00Z2025 Mar2026-06-01T19:09:25.814Z2025-06-25T03:04:33.883ZS-EPMC119548924015790210.1038/s41467-025-58259-7