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 f ive domains (A1-A2-B-C1-C2), Its catalytic domain (-B-) is flanked by dupli cated non-catalytic domains, The C-terminal repeated domains represent cell ulose-binding domains (CBDs), Xylanase domains related to the N-terminal do mains of XynA (A1-A2) are called thermostabilizing domains because their de letion normally leads to increased thermosensitivity of the enzymes. It was found that a glutathione-S-transferase (GST) hybrid protein (GST-A1A2) con taining both A-domains of XynA can interact with various soluble xylan prep arations and with mixed-linkage beta-1,3/beta-1,4-glucans, GST-A1A2 showed no affinity for insoluble microcrystalline cellulose, whereas, vice versa, GST-CP, which contains the C-terminal CBD of XynA, did not interact with so luble xylan, Another hybrid protein, GST-AS, displayed the same binding pro perties as GST-A1A2, indicating that A2 alone can also promote xylan bindin g. The dissociation constants for the binding of xylose, xylobiose, xylotri ose, xylotetraose and xylopentaose by GST-AP, 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-domain s of XynA, which are designated as xylan binding domains (XBD), are, from t he structural as well as the functional point of view, prototypes of a nove l class of binding domains. More than 50 related protein segments with hith erto unknown function were detected in about 30 other multidomain beta-glyc anases, among them putative plant (Arabidopsis thaliana) xylanases, It is a rgued that polysaccharide binding and not thermostabilization is the main f unction of A-like domains.