A novel class of protein from wheat which inhibits xylanases

Abstract

We have purified a novel class of protein that can inhibit the activity of endo-beta-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7-8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-beta-1, 4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride. The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20+/-2 mg/ml with a kcat of 103+/-6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 microM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome

“…To allow detection of eventually truncated N-and C-termini, the custom database was subsequently N-and C-terminally 'ragged' using DBToolkit prepared by spotting 1 µl of a mixture of sample and matrix (7 mg/ml CHCA in 50% ACN containing 0.1% TFA) on a stainless steel (192-well) MALDI target plate and allowed to air-dry at RT. Prior to MALDI-MS analysis, the instrument was externally calibrated with a mixture of Angiotensin I, Glu-fibrino-peptide B, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH (18-39). For MS/MS experiments, the instrument was externally calibrated with fragments of Glu-fibrino-peptide.…”

“…They occur simultaneously as a ~40 kDa single polypeptide and as a processed form existing of two disulfide-linked polypeptides of ~30 and ~10 kDa [20]. XIP-and TLXI-type XIs are basic, monomeric proteins with a molecular mass of ~30 and ~18 kDa, respectively [16,17]. Multiple putative TAXI-[18, 21, 22] and XIP-type [19,23] as well as one TLXI-type gene(s) [17] have been identified in wheat and some have been confirmed.…”

A Wheat Xylanase Inhibitor Gene,Xip-I, but NotTaxi-I, Is Significantly Induced by Biotic and Abiotic Signals That Trigger Plant Defense

“…1,2) XIP-I competitively inhibits xylanases belonging to glycoside hydrolase family 10 (GH10) and family 11 (GH11). 3) TAXI-I and TAXI-II differ in specificity for GH11 microbial xylanases.…”

Chitinases in root nodules

“…Furthermore, a conserved chitinase sequence does not necessarily denote chitinase activity. As mentioned earlier, a subgroup of class III family 18 endochitinases has no enzymatic chitinase activity and instead works as xylanase inhibitor proteins (XIPs; McLauchlan et al 1999). Xylan is the predominant hemicellulose in the plant cell walls and the second most abundant polysaccharide on earth.…”