Molecular identification of wheat endoxylanase inhibitor TAXI‐I¹, member of a new class of plant proteins

Abstract

Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in di¡erent (bio)technological processes. Here, we report on the identi¢cation of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the speci¢city and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identi¢ed class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.

Structural Basis for Inhibition of Aspergillus niger Xylanase by Triticum aestivum Xylanase Inhibitor-I

“…SDS-PAGE analysis under reducing circumstances on dissolved TAXI-I crystals indicates the presence in the crystal of both molecular forms A and B (data not shown (16). These residues are part of loop L HeCq , connecting the longest ␣-helix He present in the structure, with ␤-sheet C2, the outer rim of the C domain (Fig.…”

“…3B) TAXI-I displays significant sequence similarity with a carrot extracellular dermal glycoprotein (37), a tomato xyloglucanspecific endoglucanase inhibitor protein (38) and with TAXIlike proteins (16) identified in barley (HVXI), rye (SCXI-I to SCXI-IV), and durum wheat (TDXI-I and TDXI-II). In addition, screening of the available data bases revealed that multiple TAXI-I homologous genes also occur in soybean, rice, and Arabidopsis thaliana (16). Several amino acids in the TAXI-I sequence, notably 10 of the 12 cysteines, are generally conserved among all TAXI-I-related sequences suggesting a fundamental structural similarity.…”

“…After reduction with 2-mercaptoethanol, form B dissociates into a 10-and a 30-kDa fragment, whereas the molecular mass of form A (40 kDa) is not affected by this treatment. Because the N-terminal sequences of the 30-and 40-kDa polypeptides are identical, the 10-and 30-kDa polypeptides of form B, held together by one disulfide bond, are believed to be derived from form A by processing, possibly mediated by proteolytic enzymes (16). Xylanase inhibitor protein-I (XIP-I) is a glycosylated monomeric protein with a molecular mass of 29 kDa and a pI value of 8.7-8.9.…”

The Dual Nature of the Wheat Xylanase Protein Inhibitor XIP-I

“…The intricate recognition specificities of XIP-I toward specific xylanases from both GH10 and GH11 families might be the result of plant evolution to counteract the many xylanases secreted by bacteria and fungi, similar to polygalacturonase-inhibiting proteins (33). This could also explain why two different classes of xylanase inhibitors with different specificities and expression patterns occur in cereals (29,34). TAXI is the other type of xylanase inhibitor isolated from wheat and is specific for GH11 xylanases (35).…”

Hydrophobicversushydrophilic: ionic competition in remacemide salt structures

“…On the other hand, XIP-type inhibitors with molecular masses of 29-32 kDa, inhibit both family 10 as well as family 11 xylanases, as long as they are of fungal origin [9]. Studies on the molecular identification, isolation and characterization of the TAXI-I gene, together with screening of the available wheat EST libraries, showed clear evidence that TAXIs belong to a newly identified class of plant proteins to which a function as plant protective microbial glycoside hydrolase inhibitor can be suggested [10]. In order to study this class of proteins, their inhibition mechanics and specificities, and their influence in the above mentioned industrial processes, a crystallographic study was undertaken.…”