Characterization of the endo-beta-1,3-glucanase activity of S. cerevisiae Eng2 and other members of the GH81 family.
The GH81 family includes proteins with endo-beta-1,3-glucanase widely distributed in yeast and fungi, which are also present in plants and bacteria. We have studied the activity of the Saccharomyces cerevisiae ScEng2 and the Schizosaccharomyces pombe SpEng1 and SpEng2 proteins. All three proteins exclusively hydrolyzed linear beta-1,3-glucan chains. Laminari-oligosaccharide degradation revealed that the minimum substrate length that the three endoglucanases were able to efficiently degrade was a molecule with at least 5 glucose residues, suggesting that the active site of the enzymes recognized five glucose units. Prediction of the secondary structure of ScEng2 and comparison with proteins of known structure allowed the identification of a 404-amino acid region with a structure similar to the Clostridium thermocellum endoglucanase CelA. This fragment showed similar enzymatic characteristics to those of the complete protein, suggesting that it contains the catalytic domain of this family of proteins. Within this domain, four conserved Asp and Glu residues (D518, D588, E609, and E613) are necessary for enzymatic activity.
Martín-Cuadrado
Ana-Belén
AB
Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain.
Fontaine
Thierry
T
Esteban
Pedro-Felipe
PF
del Dedo
Javier Encinar
JE
de Medina-Redondo
Maria
M
del Rey
Francisco
F
Latgé
Jean Paul
JP
de Aldana
Carlos R Vázquez
CR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry; D017124:Conserved Sequence; D004795:Enzyme Stability; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D009844:Oligosaccharides; D004693:Glucan Endo-1,3-beta-D-Glucosidase; C074775:CelA endoglucanase; D002480:Cellulase