|
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 β‐1,3/β‐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°C by fluorescence quench experiments, were 8.1 × 10?3 M, 2.3 × 10?4 M, 2.3 × 10?5 M, 2.5 × 10?6 M and 1.1 × 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 β‐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.
|
