The role of amino‐acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda β‐glycosidase

The design of β‐glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the β‐glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzyme‐transition state complex. In an attempt to fill this gap, site‐directed mutagenesis and enzyme steady‐state kinetic experiments with different substrates were conducted, using as model a digestive β‐glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfβgly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes. Energy of noncovalent interactions in transition state between Sfβgly50 amino acids and glycone hydroxyls was determined. Sfβgly50 residue E451 seems to be a keyresidue in determining β‐glycosidase preference for glucosides vs. galactosides based on the following data: (a) The energy of the noncovalent interaction between glycone equatorial hydroxyl 4 with E451 in the transition state is about 60% higher than its interaction with Q39. (b) The energy of the E451–hydroxyl 4 interaction decreases more than the Q39–hydroxyl 4 interaction when hydroxyl 4 is changed from equatorial to axial position. (c) A Sfβgly50 mutant, where E451 was substituted by A, hydrolyzes galactosides faster than glucosides. It was also shown that glycone hydroxyl 6 interacts favorably with Q39, but not with E451, probably due to steric hindrance. These interactions result in the β‐glycosidase hydrolyzing fucosides (6‐deoxygalactosides) faster than glucosides and galactosides.