TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family.
Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by 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 expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by 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 approx. 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 thus represents a novel type of inhibitor within this group of proteins.
Fierens
Ellen
E
Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium.
Rombouts
Sigrid
S
Gebruers
Kurt
K
Goesaert
Hans
H
Brijs
Kristof
K
Beaugrand
Johnny
J
Volckaert
Guido
G
Van Campenhout
Steven
S
Proost
Paul
P
Courtin
Christophe M
CM
Delcour
Jan A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D007700:Kinetics; D013058:Mass Spectrometry; D008698:Mesylates / Q000737:chemistry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry; D013997:Time Factors; D014908:Triticum / Q000737:chemistry*; D014990:Xylans / Q000378:metabolism
D004791:Enzyme Inhibitors; D008698:Mesylates; D010940:Plant Proteins; D014990:Xylans; C003427:thaumatin protein, plant; D043364:Endo-1,4-beta Xylanases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C012077:trifluoromethanesulfonic acid