title: Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity.
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.
authors.affiliation: Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium.
authors.forename: Gert
authors.initials: G
authors.lastname: Raedschelders
authors.forename: Katleen
authors.initials: K
authors.lastname: Fierens
authors.forename: Stefaan
authors.initials: S
authors.lastname: Sansen
authors.forename: Sigrid
authors.initials: S
authors.lastname: Rombouts
authors.forename: Kurt
authors.initials: K
authors.lastname: Gebruers
authors.forename: Johan
authors.initials: J
authors.lastname: Robben
authors.forename: Anja
authors.initials: A
authors.lastname: Rabijns
authors.forename: Christophe M
authors.initials: CM
authors.lastname: Courtin
authors.forename: Jan A
authors.initials: JA
authors.lastname: Delcour
authors.forename: Steven
authors.initials: S
authors.lastname: Van Campenhout
authors.forename: Guido
authors.initials: G
authors.lastname: Volckaert
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors*; D005973:Glutamine / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011392:Proline / Q000737:chemistry; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology*; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000737:chemistry
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C422247:TAXI II protein, Triticum aestivum; D005973:Glutamine; D004247:DNA; D011392:Proline; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D043364:Endo-1,4-beta Xylanases