Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus.
We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.
Gueguen
Y
Y
Bacterial Genetics Group, Department of Microbiology, Wageningen Agricultural University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands.
Voorhorst
W G
WG
van der Oost
J
J
de Vos
W M
WM
D000595:Amino Acid Sequence; D002480:Cellulase / Q000378:metabolism; D003001:Cloning, Molecular; D005285:Fermentation; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005936:Glucans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D005936:Glucans; D011134:Polysaccharides; C008247:laminaran; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D002480:Cellulase