Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity

Raedschelders, Gert; Fierens, Katleen; Sansen, Stefaan; Rombouts, Sigrid; Gebruers, Kurt; Robben, Johan; Rabijns, A.; Courtin, Christophe M.; Delcour, Jan A.; Campenhout, Steven Van; Volckaert, Guido

doi:10.1016/j.bbrc.2005.07.103

Cited by 23 publications

(21 citation statements)

References 36 publications

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“…The characteristics displayed under such conditions may be attributed to disulphide bridging, a trait synonymous with xylanases from T. lanuginosus [27]. In the present study, incidences of varying activity between crude and purified xylanase preparations may have been mediated by proteinaceous xylanase inhibitors, common in cereal grains such as wheat bran [28,29]. During extraction of the target xylanase from fermented wheat bran, such inhibitors may have been retained within the crude xylanase preparation prior to characterisation studies.…”

Section: Discussionmentioning

confidence: 68%

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Gaffney

Carberry

Doyle

et al. 2009

Enzyme and Microbial Technology

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a b s t r a c tA xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100• C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.

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Section: Discussionmentioning

confidence: 68%

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Gaffney

Carberry

Doyle

et al. 2009

Enzyme and Microbial Technology

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“…Recent data by Raedschelders et al . [25] showed the functional importance of His374 in the interaction between TAXI‐type proteins and A. niger endoxylanase. Moreover, the TAXI combination His374/Leu292 is required for A. niger endoxylanase inhibition by TAXI‐type proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Based on structural data [16], possible candidates are the neighboring amino acid residues of TAXI‐I His374 such as Phe375 and Thr376 and the residues situated on another endoxylanase interaction TAXI‐I loop, e.g. amino acid Leu292 as shown recently for A. niger endoxylanase [25]. Determination of the crystal structures of TAXI‐I complexed with different GH11 endoxylanases and mutational analysis of enzyme and inhibitor would provide insight in the currently observed differences in enzyme‐inhibitor affinity.…”

Section: Resultsmentioning

confidence: 99%

His374 of wheat endoxylanase inhibitor TAXI‐I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

Fierens¹,

Gils

Sansen

et al. 2005

The FEBS Journal

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Wheat endoxylanase inhibitor TAXI‐I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase‐TAXI‐I complex showed His374 of TAXI‐I to be a key residue in endoxylanase inhibition [Sansen S, De Ranter CJ, Gebruers K, Brijs K, Courtin CM, Delcour JA & Rabijns A (2004) J Biol Chem 279, 36022–36028]. Its role in enzyme–inhibitor interaction was further investigated by site‐directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild‐type TAXI‐I and TAXI‐I His374 mutants were determined by surface plasmon resonance analysis. Enzyme–inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild‐type TAXI‐I towards the endoxylanases were in the range between 1.96 and 36.1 × 104m−1·s−1 and 0.72–3.60 × 10−4·s−1, respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI‐I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three‐ to 80‐fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI‐I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase–TAXI‐I complex rather than in the docking of inhibitor onto enzyme.

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“…niger xylanase complex (1T6G.pdb) reveals a direct interaction of the inhibitor with the active site region of the enzyme and further substrate-mimicking contacts with binding subsites filling the whole substrate-docking region ). The observed difference in xylanase specificity between TAXI-I and TAXI-II can be attributed to the C-terminal loop Raedschelders et al 2005;Bourgois et al 2007). In addition to the GH10 and GH11 xylanases, a GH7 enzyme XYNA from P. funiculosum was reported to be inhibited by XIP-I, TAXI-I and TAXI-II with Ki of 106, 46 and 46 nM, respectively using soluble wheat AX as substrate (Furniss et al 2005).…”

Section: Sensitivity To Proteinaceous Inhibitorsmentioning

confidence: 89%

Factors affecting xylanase functionality in the degradation of arabinoxylans

Berrin

Juge

2008

Biotechnol Lett

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Endo-beta-1,4-xylanases are key enzymes in the degradation of arabinoxylans, the main non-starch polysaccharides from grain cell walls. Due to the heterogeneity of arabinoxylans, xylanases with different characteristics are required in industrial applications but the choice of the enzyme is still largely empirical. Although the classification into glycoside hydrolase families greatly helped to derive mechanistic information on the catalytic and substrate specificity of xylanases, other factors e.g. their sensitivity to endogenous inhibitors, the presence of carbohydrate-binding module(s) and their degree of selectivity towards soluble versus insoluble substrate may play a role in determining the functionality of these enzymes in the degradation of arabinoxylans.

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Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity

Cited by 23 publications

References 36 publications

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

His374 of wheat endoxylanase inhibitor TAXI‐I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

Factors affecting xylanase functionality in the degradation of arabinoxylans

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