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Title: Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity
Authors: Raedschelders, Gert ×
Fierens, Katleen
Sansen, Stefaan
Rombouts, Sigrid
Gebruers, Kurt
Robben, Johan
Rabijns, Anja
Courtin, Christophe
Delcour, Jan
Van Campenhout, Steven
Volckaert, Guido #
Issue Date: Sep-2005
Series Title: Biochemical and Biophysical Research Communications vol:335 issue:2 pages:512-22
Abstract: Wheat grains contain Triticum aestivum xylanase inhibitor (TAXI) proteins which inhibit microbial xylanases, some of which are used in cereal based food industries. These inhibitors may play a role in plant defence. Among the TAXI isoforms described so far, TAXI-II displays a deviating inhibition specificity pattern. Here, we report on the molecular identity of TAXI-II and the basis of its inhibition specificity. Three candidate TAXI-II encoding sequences were isolated and recombinantly expressed in Pichia pastoris. To identify TAXI-II, the resulting proteins were tested against glycoside hydrolase family (GHF) 11 xylanases of Aspergillus niger (ANX) and Bacillus subtilis (BSX). One of these proteins (rTAXI-IB) inhibited both enzymes, like natural TAXI-I. The other candidates (rTAXI-IIA and rTAXI-IIB) showed an inhibition pattern typical for natural TAXI-II, only clearly inhibiting BSX. Comparative analysis of these highly similar sequences with distinct inhibition activity patterns, combined with information on the structural basis for ANX inhibition by TAXI-I [S. Sansen, C.J. De Ranter, K. Gebruers, K. Brijs, C.M. Courtin, J.A. Delcour, A. Rabijns, Structural basis for inhibition of Aspergillus niger xylanase by Triticum aestivum xylanase inhibitor-I, J. Biol. Chem. 279 (2004) 36022-36028], indicated a crucial role for Pro294 of TAXI-IIA and Gln376 of TAXI-IIB in determining the reduced inhibition activity towards ANX. Consequently, single point mutants rTAXI-IIA[P294L] and rTAXI-IIB[Q376H], both displaying the Leu/His combination corresponding to TAXI-I, were able to inhibit ANX. These results show that TAXI-II inhibition specificity bears on the identity of two key residues at positions 294 and 376, which are involved in the interaction at the -2 glycon subsite and the active site of GHF 11, respectively.
URI: 
ISSN: 0006-291X
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Division of Gene Technology (-)
Centre for Food and Microbial Technology
Biocrystallography
Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

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