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

Abstract

The design of b-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 b-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzymetransition state complex. In an attempt to fill this gap, sitedirected mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive b-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbgly50). 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 Sfbgly50 amino acids and glycone hydroxyls was determined. Sfbgly50 residue E451 seems to be a key residue in determining b-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 Sfbgly50 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 b-glycosidase hydrolyzing fucosides (6-deoxygalactosides) faster than glucosides and galactosides.Keywords: b-glucosidase; glycoside hydrolase; enzyme specificity.The b-glycosidases from glycoside hydrolases family 1 are enzymes that remove monosaccharides from the nonreducing end of di-and/or oligo-saccharides. The nonreducing monosaccharide residue binds at the glycone subsite (subsite )1), whereas the rest of the substrate molecule accommodates at the aglycone subsite, which actually may correspond to several subsites (+1, +2, +3, etc.). The family 1 comprises a total of 213 sequenced b-glycosidases, from which 8 had their tertiary structure determined [1]. According to the system of glycoside hydrolases classification [1][2][3], all b-glycosidases from family 1 present the same tertiary structure (the (b/a) 8 barrel), they are configuration-retaining glycosidases and their catalytic activity depends on two glutamic acid residues, one positioned after the b strand 4 (proton donor) and the other after the b strand 7 (nucleophile). The family 1 comprises eight different EC numbers (3.2.1.21, b-glucosidase; 3.2.1.23, b-galactosidases; 3.2.1.25, b-mannosidases; 3.2.1.85, 6-phospho-b-galactosidase; 3.2.1.86, 6-phospho-b-glucosidase; 3.2.1.62, 3.2.1.108, lactase-phlorizin hydrolase and 3.2.3.1, myrosinase) catalyzing the hydrolysis of substrates presenting a variety of glycones (monosaccharides like glucose, galactose, fucose, mannose, 6-phospho-glucose and 6-phospho-ga...

Molecular basis of substrate specificity in family 1 glycoside hydrolases

“…The combination of these data with the growing number of b-glycosidases sequences classified in the GH family 1 (1) has allowed the identification of several amino acid residues potentially involved in the substrate specificity in hundreds of b-glycosidases. Characterization of the non-covalent interactions between b-glycosidase active site and substrate (20,21) revealed that subsite 71 specificity is determined by a hydrogen bond network between glycone hydroxyls and active site residues. However, the importance of the majority of the active site residues of b-glycosidases still remains to be determined, especially those involved in the aglycone specificity.…”

Heterologous expression and enzymatic characterization of .BETA.-glucosidase from the drywood-eating termite, Neotermes koshunensis

“…Although a number of microbial b-glucosidase genes have been expressed in either E. coli, yeast or fungi (Pandey and Mishra, 1997;Saloheimo et al, 2002;Li et al, 2004;Hong et al, 2007) studies on heterologous expressions of insect b-glucosidases are still limited (Marana et al, 2002;Byeon et al, 2005).…”

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

“…The substrate subsite of ␤-glycosidases in GH family 1 contains six highly conserved amino acid residues (Gln-24, His-125, Asn-169, Trp-404, Glu-411, and Trp-412 in Abg). These conserved residues in the glycone pocket play important roles in recognizing substrates (49,50). Met-407 in AbgE358G is located in the loop between the 8th strand and the 8th helix and Glu-411, Trp-412, and Trp-404, which are components of the glycone (Ϫ1) subsite and interact with the equatorial C3-and C4-hydroxyl groups of glucose, are placed at the ends of this loop (Fig.…”