The role of amino-acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda beta-glycosidase.
The design of beta-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 beta-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 beta-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbetagly50). 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 Sfbetagly50 amino acids and glycone hydroxyls was determined. Sfbetagly50 residue E451 seems to be a key residue in determining beta-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 Sfbetagly50 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 beta-glycosidase hydrolyzing fucosides (6-deoxygalactosides) faster than glucosides and galactosides.
Marana
Sandro R
SR
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, São Paulo, 05513-970, Brazil. srmarana@iq.usp.br
Terra
Walter R
WR
Ferreira
Clélia
C
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000201:enzymology*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001617:beta-Glucosidase; D000409:Alanine