Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

Authors

  • Yael S. Balazs,

    1. Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel
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  • Elina Lisitsin,

    1. Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel
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  • Oshrat Carmiel,

    1. Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel
    Current affiliation:
    1. General R & D, Teva Pharmaceuticals Ltd, Kfar Saba, Israel
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  • Gil Shoham,

    1. Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel
    Current affiliation:
    1. Institute of Chemistry, The Hebrew University of Jerusalem, Israel
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  • Yuval Shoham,

    1. Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel
    Current affiliation:
    1. Faculty of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa, Israel
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  • Asher Schmidt

    Corresponding author
    1. Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel
    • Correspondence

      A. Schmidt, Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israel

      Fax: +972 4 829 5703

      Tel: +972 4 829 2583

      E-mail: chrschm@technion.ac.il

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Abstract

1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy (STD NMR), a comparison is made between wild-type xylanase XT6 and its acid/base catalytic mutant E159Q – a non-active, single-heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide–xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275–11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose–xylanase complexes, a ‘SASSY’ plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non-covalent interactions with H2–5 of xylobiose were observed in the wild-type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β–xylobiose anomer. The 1H solution NMR spectrum of a xylotriose–E159Q complex displays non-uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face-to-face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate-observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate-binding adjustments upon a charge change in the E159Q construct.

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