Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis

The coding sequence of the mature cyanogenic beta -glucosidase (beta -gluco side glucohydrolase, EC 3.2.1.21, linamarase) was cloned into the vector pY X243 modified to contain the SUC2 yeast secretion signal sequence and expre ssed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosy lated and showed similar stability to the plant protein. Michaelis constant s for hydrolysis of the natural substrate, linamarin (K-m = 1.06 mM) and th e synthetic p-nitrophenyl beta -D-glucopyranoside (PNP-Glc; K-m = 0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substra te complexes (pK(1)(E) = 4.4-4.8, pK(2)(E) = 6.7-7.2, pK(1)(ES), = 3.9-4.4, pK(2)(ES) = 8.3) were very similar to those of the plant enzyme. Site-dire cted mutagenesis was carried out to study the function of active-site resid ues based on a homology model generated for the enzyme using the MODELLER p rogram. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala- 201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7. 22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269As n change increased K-m for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta -glucosidase.