Katleen Fierens

Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins.

riticum estivum endo ylanase nhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI‐I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI‐I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI‐I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.

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

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.