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

Abstract

β-Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities [Henrissat, B. and Bairoch, A. (1993) Biochem. J. 293, 781-788]. The β-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, U. and Withers, S.O.(1994) Anal. Biochem. 212, 259-262]. Thermoanaerobacterium saccharolyticum B6A-RI β-xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p-nitrophenyl β-D-xylopyranoside was followed by 1H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J ~ 10 Hz) assignable to H1 of free β-xylose with a small amount of α-xylose (5.05 ppm, J ~ 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticum β-xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of β-xylosidases, suggests that these two families have major differences in their active-site geometrics. Consistent with its retaining mechanism, β-xylosidase of T. saccharolyticum B6A-RI also displayed transglycosylating activity: reverse-phase HPLC showed approximately 30% conversion of p-nitrophenyl-β-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 β-1,4, β-1,3- and β-1,2-linked xylo-oligosaccharides.