Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10ACD) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBDX) or fused to the catalytic domain (Pf Xyn10A′), bound to amorphous and bacterial microcrystalline cellulose with a Ka of 2.5×105 M-1. CBDX exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A′ was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10ACD; however, Pf Xyn10A′ and Pf Xyn10ACD exhibited the same activity against soluble substrates. CBDX did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10ACD when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A′, and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10ACD via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBDX is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase.
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September 1999
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Research Article|
August 24 1999
The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism
Jaitinder GILL;
Jaitinder GILL
*Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
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Jane E. RIXON;
Jane E. RIXON
*Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
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David N. BOLAM;
David N. BOLAM
*Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
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Simon MCQUEEN-MASON;
Simon MCQUEEN-MASON
†The Plant Laboratory, Department of Botany, University of York, Heslington, York YO1 5DD, U.K.
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Peter J. SIMPSON;
Peter J. SIMPSON
‡Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, U.K.
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Michael P. WILLIAMSON;
Michael P. WILLIAMSON
‡Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, U.K.
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Geoffrey P. HAZLEWOOD;
Geoffrey P. HAZLEWOOD
§Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, U.K.
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Harry J. GILBERT
Harry J. GILBERT
1
*Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
1To whom correspondence should be addressed (H.Gilbert@Newcastle.ac.uk).
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Publisher: Portland Press Ltd
Received:
April 08 1999
Revision Received:
May 19 1999
Accepted:
July 05 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (2): 473–480.
Article history
Received:
April 08 1999
Revision Received:
May 19 1999
Accepted:
July 05 1999
Citation
Jaitinder GILL, Jane E. RIXON, David N. BOLAM, Simon MCQUEEN-MASON, Peter J. SIMPSON, Michael P. WILLIAMSON, Geoffrey P. HAZLEWOOD, Harry J. GILBERT; The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism. Biochem J 1 September 1999; 342 (2): 473–480. doi: https://doi.org/10.1042/bj3420473
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