Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: Nmr assignments, pH titration, and hydrogen exchange

Authors

  • Leigh A. Plesniak,

    1. Department of Biochemistry and Molecular Biology, Department of Chemistry, and the Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    Current affiliation:
    1. Chemistry Department, University of San Diego, San Diego, California 92110
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  • Gregory P. Connelly,

    1. Department of Biochemistry and Molecular Biology, Department of Chemistry, and the Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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  • Lawrence P. Mcintosh,

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, Department of Chemistry, and the Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    • Department of Biochemistry and Molecular Biology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3;
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  • Warren W. Wakarchuk

    1. Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6
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Abstract

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1Hϕ2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 × 10−5 s−1 at pH* 7.04 and 30°C) is retarded by > 106-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at ˜6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral Nϕ2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30°C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.

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