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Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding

Lookup NU author(s): Carl Morland, Professor Anthony O'Donnell, Emeritus Professor Harry Gilbert


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Crystal structure analysis of Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) indicated that the enzyme contained a single calcium binding site that did not exhibit structural features typical of the EF-hand motif. Isothermal titration calorimetry revealed that XYLA binds calcium with a K(a) of 4.9 x 104 M-1 and a stoichiometry consistent with one calcium binding site per molecule of enzyme. Occupancy of the calcium binding domain with its ligand protected XYLA from proteinase and thermal inactivation and increased the melting temperature of the enzyme from 60.8 to 66.5 °C. However, the addition of calcium or EDTA did not influence the catalytic activity of the xylanase. Replacement of the calcium binding domain, which is located within loop 7 of XYLA, with the corresponding short loop from Cex (a Cellulomonas fimi xylanase/exoglucanase), did not significantly alter the biochemical properties of the enzyme. These data suggest that the primary function of the calcium binding domain is to increase the stability of the enzyme against thermal unfolding and proteolytic attack. To understand further the nature of the calcium binding domain of XYLA, four variants of the xylanase, D256A, N261A, D262A, and XYLA''', in which Asp-256, Asn-261, and Asp-262 had all been changed to alanine, were constructed. These mutated enzymes did not show any significant binding to Ca2+, indicating that Asp- 256, Ass-261, and Asp-262 play a pivotal role in the affinity of XYLA for the divalent cation. In the presence or absence of calcium, XYLA'' exhibited thermal stability similar to that of the native enzyme bound to Ca2+ ions, although the variant was sensitive to proteinase inactivation. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed.

Publication metadata

Author(s): Spurway TD, Morland C, Cooper A, Sumner I, Hazlewood GP, O'Donnell AG, Pickersgill RW, Gilbert HJ

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 1997

Volume: 272

Issue: 28

Pages: 17523-17530

Print publication date: 11/07/1997

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology, Inc.


DOI: 10.1074/jbc.272.28.17523

PubMed id: 9211898


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