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

The coding sequence of the mature cyanogenic β-glucosidase (β-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae . The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin ( K m = 1.06mM) and the synthetic p -nitrophenyl β-D-glucopyranoside (PNP-Glc; K m = 0.36mM), as well as apparent p K a values of the free enzyme and the enzymeŐsubstrate complexes (p K E 1 = 4.4Ő4.8, p K E 2 = 6.7Ő7.2, p K ES 1 = 3.9Ő4.4, p K ES 2 = 8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. 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 p K a of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K m for linamarin hydrolysis 16-fold (16.1mM) and that for PNP-Glc only 2.5-fold (0.84mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava β-glucosidase.