Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity.
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.
Raedschelders
Gert
G
Laboratory of Gene Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium.
Fierens
Katleen
K
Sansen
Stefaan
S
Rombouts
Sigrid
S
Gebruers
Kurt
K
Robben
Johan
J
Rabijns
Anja
A
Courtin
Christophe M
CM
Delcour
Jan A
JA
Van Campenhout
Steven
S
Volckaert
Guido
G
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
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