Luang, S.Fernández-Luengo, X.Nin-Hill, A.Streltsov, V.A.Schwerdt, J.G.Alonso-Gil, S.Ketudat Cairns, J.R.Pradeau, S.Fort, S.Maréchal, J.-D.Masgrau, L.Rovira, C.Hrmova, M.2022-09-262022-09-262022Nature Communications, 2022; 13(1):5577-1-5577-192041-17232041-1723https://hdl.handle.net/2440/136479In the barley β-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures β-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multiscale molecular modelling methods to examine the binding and conformational behaviour of isomeric β-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-β-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutationspecific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.en© Crown 2022 This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.PlantsGlycoside HydrolasesGlucosidasesGlycosidesGlucosidesGlucoseTryptophanCrystallography, X-RaySubstrate SpecificityKineticsJournal article10.1038/s41467-022-33180-52022-09-24622382Schwerdt, J.G. [0000-0002-0476-031X]Hrmova, M. [0000-0002-3545-0605]