Abstract
Recently, we isolated and cloned two xyloglucan-specific endoglucanases (XEGs) from Geotrichum sp. M 128 and Gram-positive bacterium sp. KM 21, and a xyloglucan-specific exoglucanase, oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) from Geotrichum sp. M 128, all of which belong to glycoside hydrolase family 74. Geotrichum and KM 21 XEGs have endoglucanase activity toward xyloglucan but not cellulose, while Geotrichum OXG-RCBH is a unique exoglucanase that releases two Glc residue segments from the reducing end of the xyloglucan main chain. Further analysis of the substrate specificities of Geotrichum XEG and OXG-RCBH using various oligosaccharides revealed that they recognize specific xylose branching structures. Although both Geotrichum XEG and OXG-RCBH have at least four subsites (-2 to +2), specific recognition of xylose residues occurs at the +1 and +2 sites in the former and at the -1 site in the latter. Moreover, xylose branching at the reducing end (site +2) eliminates OXG-RCBH activity. This enzymatic activity is very different from those of known glycosidases. Recently, OXG-RCBH was assigned a new EC number (EC 3.2.1.150). To elucidate the molecular mechanism of OXG-RCBH, its three-dimensional structure was analyzed. The X-ray crystal structure of OXG-RCBH revealed a unique feature of this enzyme: OXG-RCBH consists of two similar domains, both of which are folded into seven-bladed β-propeller structures. The cleft between the two domains can accommodate the oligosaccharide substrate and thus constitutes a putative catalytic core. Mutation of either Asp 35 or Asp 465, located in the cleft, to Asn resulted in a protein with no detectable catalytic activity, indicating the critical role of these amino acids in catalysis.
