His374 of wheat endoxylanase inhibitor TAXI‐I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

• Published in: The FEBS journal 2005-11, Vol.272 (22), p.5872-5882
• Main Authors: Fierens, Katleen, Gils, Ann, Sansen, Stefaan, Brijs, Kristof, Courtin, Christophe M., Declerck, Paul J., De Ranter, Camiel J., Gebruers, Kurt, Rabijns, Anja, Robben, Johan, Campenhout, Steven, Volckaert, Guido, Delcour, Jan A.
• Format: Article
• Language: English
• Subjects: Alanine - metabolism; Amino Acid Substitution; Aspergillus niger; Aspergillus niger - enzymology; Bacillus subtilis; Bacillus subtilis - enzymology; Binding sites; Circular Dichroism; Endo-1,4-beta Xylanases - antagonists & inhibitors; Endo-1,4-beta Xylanases - classification; Endo-1,4-beta Xylanases - metabolism; endoxylanase; Enzymes; Glutamine - metabolism; Histidine - chemistry; Hydrogen Bonding; Hydrogen-Ion Concentration; inhibitor; Inhibitor drugs; Isoelectric Focusing; Kinetics; Lysine - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Pichia - metabolism; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Proteins - pharmacology; Protein Binding; protein–protein interaction; Recombinant Proteins - metabolism; Surface Plasmon Resonance; TAXI; Trichoderma; Trichoderma - enzymology; Trichoderma longibrachiatum; Triticum - enzymology; Triticum aestivum; Wheat
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2005.04987.x

Wheat endoxylanase inhibitor TAXI‐I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase‐TAXI‐I complex showed His374 of TAXI‐I to be a key residue in endoxylanase inhibition [Sansen S, De Ranter CJ, Gebruers K, Brijs K, Courtin CM, Delcour JA & Rabijns A (2004) J Biol Chem 279, 36022–36028]. Its role in enzyme–inhibitor interaction was further investigated by site‐directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild‐type TAXI‐I and TAXI‐I His374 mutants were determined by surface plasmon resonance analysis. Enzyme–inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild‐type TAXI‐I towards the endoxylanases were in the range between 1.96 and 36.1 × 104m−1·s−1 and 0.72–3.60 × 10−4·s−1, respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI‐I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three‐ to 80‐fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI‐I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase–TAXI‐I complex rather than in the docking of inhibitor onto enzyme.