Purification and Partial Characterization of an Endoxylanase Inhibitor from Barley

Goesaert, Hans; Debyser, Winok; Gebruers, Kurt; Proost, Paul; Damme, Jozef Van; Delcour, Jan A.

doi:10.1094/cchem.2001.78.4.453

Cited by 46 publications

(45 citation statements)

References 27 publications

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“…The N-terminal amino acid sequences of TAXI-I and TAXI-II showed a high degree of identity, but there was no similarity to XIP-I. The TAXI-like inhibitors are believed to be active against bacterial and fungal family 11 endoxylanases but not against family 10 endoxylanases (46,47). XIP-I inhibited both family-10 and 11 fungal xylanases apart from the family 10 Aspergillus aculeatus xylanase with K i values ranging from 3.4 to 610 nM, but bacterial family 10 and 11 xylanases were not inhibited (48).…”

mentioning

confidence: 99%

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Tahir

Berrin

Flatman

et al. 2002

Journal of Biological Chemistry

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The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k cat and K m , respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K m and decreased k cat . Tyr 6 , Tyr 10 , Tyr 89 , Tyr 164 , and Trp 172 are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the threedimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases.

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mentioning

confidence: 99%

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Tahir

Berrin

Flatman

et al. 2002

Journal of Biological Chemistry

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“…Its deduced amino acid sequence corresponded well with the experimentally derived sequence of the SCXI-I 10 kDa polypeptide (21 residues, Goesaert et al 2002). A 3 0 RACE reaction on total RNA isolated from S. cereale cultivar Halo immature ears was performed using the TAXI-I Fig. 3 Alignment at the amino acid level of the identified TAXI-I, HVXI and SCXI-II/III coding sequences (Clustal W, EBI; using default parameters) matching experimentally derived peptides (underlined) Goesaert et al 2001Goesaert et al , 2002. The signal sequence is indicated in bold.…”

Section: Identification Of Scxi Genes In Secale Cerealementioning

confidence: 61%

“…For the identification of the TAXI-like genes in barley and rye, the same varieties were used as for the biochemical characterization of the TAXI-like proteins in these cereals by Goesaert et al (2001Goesaert et al ( , 2002, namely barley (H. vulgare) cultivar Hiro and rye (S. cereale) cultivar Halo.…”

Section: Plant Materialsmentioning

confidence: 99%

Molecular identification and chromosomal localization of genes encoding Triticum aestivum xylanase inhibitor I-like proteins in cereals

et al. 2004

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TAXI ( Triticum aestivum xylanase inhibitor) proteins are present in wheat flour and are known to inhibit glycosyl hydrolase family 11 endoxylanases, enzymes which are commonly applied in grain processing. Here, we describe the PCR-based molecular identification of genes encoding endoxylanase inhibitors HVXI and SCXI, the TAXI-like proteins from barley ( Hordeum vulgare) and rye ( Secale cereale) respectively. The HVXI coding sequence encodes a mature protein of 384 amino acids preceded by a 19 amino acid long signal sequence. SCXI-II/III has an open reading frame encoding a signal peptide of 21 amino acids and a mature protein of 375 amino acids. As for TAXI-I, no introns were detected in the untranslated regions and coding sequences identified. These newly identified sequences allowed us to perform a multiple sequence alignment with TAXI-I and similar proteins. Rice TAXI-type proteins clustered together with the cereal endoxylanase inhibitors. Dicotyledonous proteins with sequence similarity to TAXI-I, including the tomato xyloglucan-specific endoglucanase inhibiting protein, formed a different clade. The TAXI-type proteins may hence be part of a superfamily of proteins all involved in plant responses to biotic or abiotic stress and for which a function as glycosyl hydrolase inhibitors can be suggested. The chromosomal localization of the TAXI-I gene identified on wheat chromosome 3B, of the SCXI-II/III gene identified on rye chromosome 6R, and the presence of a cluster of TAXI-like genes on rice chromosome 1, allowed us to assign the location of TAXI-like genes to the wheat-rye translocation area 3BL/6RL characterized by RFLP markers XGlb33 and Xpsr454 and isozyme Est-5. In rice, RFLP marker C1310S corresponds to a TAXI-like protein encoding sequence.

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“…Debyser et al (1999) purified this inhibitor from wheat flour and gave the name TAXI (for Triticum aestivum xylanase inhibitor). However, later on TAXI type inhibitors were purified from barley (Hordeum vulgare xylanase inhibitor, HVXI) (Goesaert et al, 2001), Durum wheat (Triticum durum xylanase inhibitor, TDXI) (Goesaert et al, 2003b) and Rye (Secale cereale xylanase inhibitor, SCXI) (Goesaert, 2002). Approximate levels of TAXI type xylanase inhibitors in these cereals are presented in Table 2.4.…”

Section: Xylanase Inhibitorsmentioning

confidence: 99%

“…Bacillus subtilis xylanase XIP inhibits both GH 10 and GH 11 xylanases, but only those of fungal origin (Dornez et al, 2009). On the other hand, TAXI type inhibitors inhibit xylanases of fungal and bacterial origin from GH family 11 and not GH family 10 (Goesaert et al, 2001). This understanding about the inhibition specificity of the xylanase inhibitors has been exploited to interpret the amount of each inhibitor in the presence of each other.…”

Section: Xylanase Inhibitorsmentioning

confidence: 99%

Variability in arabinoxylan, xylanase activity, and xylanase inhibitor levels in hard spring wheat

Mendis¹,

Delcour²,

Gebruers³

et al. 2012

CFW Plexus

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Arabinoxylans (AX), xylanase, and xylanase inhibitors have an important role in many cereal food processing applications. The effect of growing location (L), genotype (G), and their interactions (L*G) on AX, apparent xylanase and apparent xylanase inhibition activities of Triticum aestivum xylanase inhibitor (TAXI) and xylanases inhibiting protein (XIP) of wheat was studied using six hard red spring wheat and six hard white spring wheat genotypes. Total AX content was largely dependent on L*G. In bran, xylanase activity, 25% of the variability could be attributed to L*G interaction. Genotype contributed 72% to the variability in TAXI activity indicating TAXI activity in wheat bran is largely under genetic control. The bran TAXI level and XIP level varied by a factor of 700 and 3000, respectively, among genotypes. These results enable the industry to choose between different wheat varieties with varying xylanase activities to complement its intended use.iv ACKNOWLEDGEMENTS

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Purification and Partial Characterization of an Endoxylanase Inhibitor from Barley

Cited by 46 publications

References 27 publications

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Molecular identification and chromosomal localization of genes encoding Triticum aestivum xylanase inhibitor I-like proteins in cereals

Variability in arabinoxylan, xylanase activity, and xylanase inhibitor levels in hard spring wheat

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