Stereochemical course and reaction products of the action of beta-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI

beta-Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities [Henrissat, B. & Bairoch, A. (1993) Biochem. J. 293, 781-788]. The beta-xylosidase from Butyrivibrio fibrisolvens, which belongs to family 43, has been shown to operate by a molecular mechanism which results in the inversion of the anomeric configuration [Braun, C., Meinke, A., Ziser, L. & Withers, S. G. (1994) Anal. Biochem. 212, 259-262]. Thermoanaerobacterium saccharolyticum EGA-RI beta-xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xylopyranoside was followed by H-1 NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J approximate to 10 Hz) assignable to H1 of free beta-xylose with a small amount of alpha-xylose (5.05 ppm, J approximate to 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticum beta-xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of beta-xylosidases, suggests that these two families have major differences in their active-site geometries. Consistent with its retaining mechanism, beta-xylosidase of T. saccharolyticum EGA-RI also displayed transglycosylating activity: reverse-phase HPLC showed approximately 30% conversion of p-nitrophenyl beta-D-xylopyranoside into a number of higher nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determined by total correlation spectroscopy NMR and showed that the enzyme can build beta-1,4, beta-1,3- and beta-1,2-linked xylo-oligosaccharides.