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 classi fication of glycosyl hydrolases based on amino acid sequence similarit ies [Henrissat, B. & Bairoch, A. (1993) Biochem. J. 293, 781-788]. The beta-xylosidase from Butyrivibrio fibrisolvens, which belongs to fami ly 43, has been shown to operate by a molecular mechanism which result s in the inversion of the anomeric configuration [Braun, C., Meinke, A ., Ziser, L. & Withers, S. G. (1994) Anal. Biochem. 212, 259-262]. The rmoanaerobacterium saccharolyticum EGA-RI beta-xylosidase which belong s to family 39 was purified as a recombinant enzyme from Escherichia c oli. The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xyl opyranoside was followed by H-1 NMR. The spectrum recorded after 2 h h ydrolysis 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 alp ha-xylose (5.05 ppm, J approximate to 3 Hz) attributable to mutarotati on. This result indicates that T. saccharolyticum beta-xylosidase oper ates with overall retention of the anomeric configuration. This result , with the lack of sequence similarity between the two families of bet a-xylosidases, suggests that these two families have major differences in their active-site geometries. Consistent with its retaining mechan ism, beta-xylosidase of T. saccharolyticum EGA-RI also displayed trans glycosylating activity: reverse-phase HPLC showed approximately 30% co nversion of p-nitrophenyl beta-D-xylopyranoside into a number of highe r nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determin ed by total correlation spectroscopy NMR and showed that the enzyme ca n build beta-1,4, beta-1,3- and beta-1,2-linked xylo-oligosaccharides.