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

Abstract

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.

Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display

“…These hypotheses based on the combinatorial exploration of the thumb region and accompanying sequence analyses were confirmed experimentally by testing the inhibitor sensitivity of a limited set of recombinantly expressed XynII mutants. The finding that Asn at position 124 of XynII is critical for XIP-I-sensitivity of this GH11 endoxylanase agrees with data obtained for another GH11 endoxylanase, ExlA from A. niger, as mutation of the corresponding Asn to Ala (ExlA-N117A) led to abolition of ExlA-XIP-I complex formation (Tahir et al, 2002). Furthermore, our results are consistent with the recently reported crystal structures of XIP-I and TAXI-I in complex with GH11 endoxylanase XynC of P. funiculosum and ExlA (Sansen et al, 2004), respectively.…”

“…To address this question, we applied the previously developed phage display system to explore the potential for diversification of the inhibition profile of GH11 endoxylanases (Table 2) by screening a combinatorial library. The library was focussed on the 'thumb' region of GH11 endoxylanases, as Aspergillus niger endoxylanase ExlA lost XIP-I sensitivity upon site-directed mutagenesis in its thumb region (Tahir et al, 2002). The crystal structure of endoxylanase-inhibitor complexes confirmed that the thumb region indeed constitutes an important interaction site not only for XIP-type inhibitors but also for TAXI-type inhibitors (Sansen et al, 2004).…”

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

“…Inhibition of fungal xylanases is not due to the binding of XIP-I to glycodecorations, because both the native and Escherichia coli-derived recombinant forms of P. funiculosum xylanase are inhibited to the same extent. Recent site-directed mutagenesis studies have shown that XIP-I binds to the conserved "thumb" hairpin loop of GH11 xylanases (13); however, the mechanism by which the inhibitor recognizes these structures and how the plant protein interacts with GH10 enzymes is unknown.…”

Endo-1,4-β-xylanase B from Aspergillus cf. niger BCC14405 Isolated in Thailand: Purification, Characterization and Gene Isolation

“…Primer sequences are boxed. Tahir et al, 2002). Based on structural analysis, both endoxylanase A and B belong to family 11.…”