Comparison of a beta-glucosidase and a beta-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Purification, characterization, gene cloning, and sequence analysis.
Two distinct exo-acting, beta-specific glycosyl hydrolases were purified to homogeneity from crude cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus: a beta-glucosidase, corresponding to the one previously purified by Kengen et al. (Kengen, S. W. M., Luesink, E. J., Stams, A. J. M., and Zehnder, A. J. B. (1993) Eur. J. Biochem. 213, 305-312), and a beta-mannosidase. The beta-mannosidase and beta-glucosidase genes were isolated from a genomic library by expression screening. The nucleotide sequences predicted polypeptides with 510 and 472 amino acids corresponding to calculated molecular masses of 59.0 and 54.6 kDa for the beta-mannosidase and the beta-glucosidase, respectively. The beta-glucosidase gene was identical to that reported by Voorhorst et al. (Voorhorst, W. G. B., Eggen, R. I. L., Luesink, E. J., and deVos, W. M. (1995) J. Bacteriol. 177, 7105-7111; GenBank accession no. U37557U37557). The deduced amino acid sequences showed homology both with each other (46.5% identical) and with several other glycosyl hydrolases, including the beta-glycosidases from Sulfolobus solfataricus, Thermotoga maritima, and Caldocellum saccharolyticum. Based on these sequence similarities, the beta-mannosidase and the beta-glucosidase can both be classified as family 1 glycosyl hydrolases. In addition, the beta-mannosidase and beta-glucosidase from P. furiosus both contained the conserved active site residues found in all family 1 enzymes. The beta-mannosidase showed optimal activity at pH 7.4 and 105 degrees C. Although the enzyme had a half-life of greater than 60 h at 90 degrees C, it is much less thermostable than the beta-glucosidase, which had a reported half-life of 85 h at 100 degrees C. Km and Vmax values for the beta-mannosidase were determined to be 0.79 mM and 31.1 micromol para-nitrophenol released/min/mg with p-nitrophenyl-beta-D-mannopyranoside as substrate. The catalytic efficiency of the beta-mannosidase was significantly lower than that reported for the P. furiosus beta-glucosidase (5.3 versus 4, 500 s-1 mM-1 with p-nitrophenyl-beta-D-glucopyranoside as substrate). The kinetic differences between the two enzymes suggest that, unlike the beta-glucosidase, the primary role of the beta-mannosidase may not be disaccharide hydrolysis. Other possible roles for this enzyme are discussed.
Bauer
M W
MW
Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Bylina
E J
EJ
Swanson
R V
RV
Kelly
R M
RM
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D005798:Genes, Bacterial; D007526:Isoelectric Point; D007700:Kinetics; D008361:Mannosidases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification / Q000378:metabolism*; D044902:beta-Mannosidase
D008361:Mannosidases; D001617:beta-Glucosidase; D044902:beta-Mannosidase