tailieunhanh - Báo cáo khoa học: Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis

The specificity of the Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) for fucosides, glucosides and galactosides is determined by noncovalent interactions of glycone6-OHandglycone 4-OHwith the active-site residues kinetics were described, showing that replacement of E451 with glutamine increased the preference of Sfbgly50 for glucosides in comparison to galactosides, whereas replacing E451 with serine had the opposite effect. | Eur. J. Biochem. 271 4169-4177 2004 FEBS 2004 doi Investigation of the substrate specificity of a b-glycosidase from Spodoptera frugiperda using site-directed mutagenesis and bioenergetics analysis Sandro R. Marana Eduardo H. P. Andrade Clelia Ferreira and Walter R. Terra Departamento de Bioquimica Instituto de Quimica Universidade de Scio Paulo Scio Paulo Brazil The specificity of the Spodoptera frugiperda digestive b-glycosidase Sfbgly50 for fucosides glucosides and galactosides is determined by noncovalent interactions of glycone 6-OH and glycone 4-OH with the active-site residues Q39 and E451. Site-directed mutagenesis and enzyme steadystate kinetics were described showing that replacement of E451 with glutamine increased the preference of Sfbgly50 for glucosides in comparison to galactosides whereas replacing E451 with serine had the opposite effect. In contrast the replacement of E451 with aspartate did not change Sfbgly50 specificity. The energy of the interactions formed by these different residues with the axial and equatorial glycone 4-OH were also measured showing that the increase in preference for galactosides resulted from a larger energy decrease in the interaction with equatorial 4-OH than with axial 4-OH vs. kJ-mol-1 whereas the increase in preference for glucosides was caused by an energy reduction in the interaction with the axial 4-OH kJ-mol-1 . The introduction of glutamine at position 451 or of asparagine at position 39 increased the preference of Sfbgly50 for fucosides in comparison to galactosides whereas the presence of aspartate or serine at position 451 had less effect on this preference. The hydrolysis of fucosides was favored because glutamine at position 451 increased a steric hindrance with 6-OH of kJ-mol-1 and asparagine at position 39 disrupted a favorable interaction with this same hydroxyl. In conclusion it is proposed that the specificity of new b-glycosidase mutants can be .