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Lookup NU author(s): Dr David Bolam, Emeritus Professor Harry Gilbert
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Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknown function (termed 'X6' modules) that had been implicated in thermostability. We have investigated the properties of two such 'thermostabilizing' modules, X6a and X6b from the Clostridium thermocellum xylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined. The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 μM for xylotriose; and 50 μM for xylobiose. Negative Δd and ΔS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three- dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 Å. The protein is a β-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re- assigned as family 22 carbohydrate-binding modules.
Author(s): Charnock S, Bolam DN, Turkenburg J, Gilbert HJ, Ferreira L, Davies G, Fontes C
Publication type: Article
Publication status: Published
Journal: Biochemistry
Year: 2000
Volume: 39
Issue: 17
Pages: 5013-5021
ISSN (print): 0006-2960
ISSN (electronic): 1943-295X
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/bi992821q
DOI: 10.1021/bi992821q
PubMed id: 10819965
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