The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage beta-1,3/beta-1, 4-glucan.
Thermotoga maritima XynA is an extremely thermostable modular enzyme with five domains (A1-A2-B-C1-C2). Its catalytic domain (-B-) is flanked by duplicated non-catalytic domains. The C-terminal repeated domains represent cellulose-binding domains (CBDs). Xylanase domains related to the N-terminal domains of XynA (A1-A2) are called thermostabilizing domains because their deletion normally leads to increased thermosensitivity of the enzymes. It was found that a glutathione-S-transferase (GST) hybrid protein (GST-A1A2) containing both A-domains of XynA can interact with various soluble xylan preparations and with mixed-linkage beta-1,3/beta-1,4-glucans. GST-A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST-C2, which contains the C-terminal CBD of XynA, did not interact with soluble xylan. Another hybrid protein, GST-A2, displayed the same binding properties as GST-A1A2, indicating that A2 alone can also promote xylan binding. The dissociation constants for the binding of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose by GST-A2, as determined at 20 degrees C by fluorescence quench experiments, were 8.1 x 10(-3) M, 2.3 x 10(-4) M, 2.3 x 10(-5) M, 2.5 x 10(-6)M and 1.1 x 10(-6) M respectively. The A-domains of XynA, which are designated as xylan binding domains (XBD), are, from the structural as well as the functional point of view, prototypes of a novel class of binding domains. More than 50 related protein segments with hitherto unknown function were detected in about 30 other multidomain beta-glycanases, among them putative plant (Arabidopsis thaliana) xylanases. It is argued that polysaccharide binding and not thermostabilization is the main function of A-like domains.
Meissner
K
K
Institut für Mikrobiologie und Genetik, Georg-August-Universität, Göttingen, Germany.
Wassenberg
D
D
Liebl
W
W
D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014990:Xylans / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D047071:beta-Glucans
D005936:Glucans; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014990:Xylans; D047071:beta-Glucans; C067858:beta-glucan, (1-3)(1-4)-; D002482:Cellulose; D005982:Glutathione Transferase; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases