Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat

Abstract

We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem. J. 338, 441-446]. In the present study, we show that XIP-I inhibits family-10 and -11 fungal xylanases. The K(i) values for fungal xylanases ranged from 3.4 to 610 nM, but bacterial family-10 and -11 xylanases were not inhibited. Unlike many glycosidase inhibitors, XIP-I was not a slow-binding inhibitor of the Aspergillus niger xylanase. Isothermal titration calorimetry of the XIP-I-A. niger xylanase complex showed the formation of a stoichiometric (1:1) complex with a heat capacity change of -1.38 kJ x mol(-1) x K(-1), leading to a predicted buried surface area of approx. 2200+/-500 A(2) at the complex interface. For this complex with A. niger xylanase (K(i)=320 nM at pH 5.5), titration curves indicated that an observable interaction occurred at pH 4-7, and this was consistent with the pH profile of inhibition of activity. In contrast, the stronger complex between A. nidulans xylanase and XIP-I (K(i)=9 nM) led to an observable interaction across the entire pH range tested (3-9). Using surface plasmon resonance, we show that the differences in the binding affinity of XIP-I for A. niger and A. nidulans xylanase are due to a 200-fold lower dissociation rate k(off) for the latter, with only a small difference in association rate k(on).

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

“…The chosen strategy for mutational analysis of A. niger xylanase took advantage of data available on the A. niger xylanase-XIP-I complex such as (a) kinetics of inhibition, (b) titration curves, (c) isothermal calorimetry data, and also the availability of (d) the three-dimensional structure of A. niger xylanase (Protein Data Bank code 1UKR) and (e) an efficient heterologous system for expression of A. niger xylanase. The inhibition mechanism of XIP-I against family 11 fungal A. niger xylanase has been studied in detail (48). The inhibition is pH-dependent in the range of 4 to 7 as determined by activity assays and titration curves, illustrating the importance of electrostatic interactions in the strength of the interaction.…”

“…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). We have previously reported the production and characterization of the A. niger xylanase in Pichia pastoris and shown that the recombinant enzyme was similar to the native enzyme and was competitively inhibited by XIP-I with a K i ϭ 350 nM (33,48). The chosen strategy for mutational analysis of A. niger xylanase took advantage of data available on the A. niger xylanase-XIP-I complex such as (a) kinetics of inhibition, (b) titration curves, (c) isothermal calorimetry data, and also the availability of (d) the three-dimensional structure of A. niger xylanase (Protein Data Bank code 1UKR) and (e) an efficient heterologous system for expression of A. niger xylanase.…”

“…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). We have previously reported the production and characterization of the A. niger xylanase in Pichia pastoris and shown that the recombinant enzyme was similar to the native enzyme and was competitively inhibited by XIP-I with a K i ϭ 350 nM (33,48).…”

“…The inhibition is pH-dependent in the range of 4 to 7 as determined by activity assays and titration curves, illustrating the importance of electrostatic interactions in the strength of the interaction. Moreover, isothermal titration calorimetry of the XIP-I:A. niger xylanase complex showed the formation of a complex with a stoichiometry of (1:1) and a heat capacity change of Ϫ1.38 kJ/mol, suggesting that the interaction was enthalpy-driven (48). Aromatic and charged amino acids are believed to play a pivotal role in protein-protein interactions by forming hydrophobic stacking and electrostatic interactions, respectively, with the target ligand.…”

Hydrophobicversushydrophilic: ionic competition in remacemide salt structures

“…While TAXI-I inhibition activity seems to be independent of the pH optima of xylanases, TAXI-II inhibition of xylanases with low pH optima is weak or absent [8]. 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].…”