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. 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. Thermoanaerobacterium saccharolyticum B6A-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 1H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J approximately 10 Hz) assignable to H1 of free Beta-xylose with a small amount of alpha-xylose (5.05 ppm, J approximately 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 B6A-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.
Armand
S
S
Centre de Recherches sur les Macromolécules Végétales, CNRS, Grenoble,France.
Vieille
C
C
Gey
C
C
Heyraud
A
A
Zeikus
J G
JG
Henrissat
B
B
D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D002240:Carbohydrate Sequence; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013237:Stereoisomerism; D014995:Xylosidases / Q000378:metabolism*
D011994:Recombinant Proteins; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase