TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family
Résumé
Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described here for the first time. Based on its up to 60 % similarity with thaumatin-like proteins (TLPs) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as thaumatin-ike xylanase inhibitor (TLXI). TLXI is a basic (pI > 9.3 in isoelectric focusing) protein with a molecular mass of approximately 18 kDa (SDS-PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26 amino acid signal sequence followed by a 151 amino acid mature protein with a calculated molecular mass of 15.6 kDa and pI of 8.38. The mature TLXI protein was successfully expressed in Pichia pastoris resulting in a 21 kDa (SDS-PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow-tight binding inhibitor, with a K i} of approximately 60 nM. Except for zeamatin, an {alpha}-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI hence represents a novel type of inhibitor within this group of proteins.
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