Investigation of the substrate specificity of a β‐glycosidase from Spodoptera frugiperda using site‐directed mutagenesis and bioenergetics analysis

Abstract

The specificity of the Spodoptera frugiperda digestive b-glycosidase (Sfbgly50) for fucosides, glucosides and galactosides is determined by noncovalent interactions of glycone 6-OH and glycone 4-OH with the active-site residues Q39 and E451. Site-directed mutagenesis and enzyme steadystate kinetics were described, showing that replacement of E451 with glutamine increased the preference of Sfbgly50 for glucosides in comparison to galactosides, whereas replacing E451 with serine had the opposite effect. In contrast, the replacement of E451 with aspartate did not change Sfbgly50 specificity. The energy of the interactions formed by these different residues with the axial and equatorial glycone 4-OH were also measured, showing that the increase in preference for galactosides resulted from a larger energy decrease in the interaction with equatorial 4-OH than with axial 4-OH (22.6 vs. 13.9 kJAEmol ). The introduction of glutamine at position 451 or of asparagine at position 39 increased the preference of Sfbgly50 for fucosides in comparison to galactosides, whereas the presence of aspartate or serine at position 451 had less effect on this preference. The hydrolysis of fucosides was favored because glutamine at position 451 increased a steric hindrance with 6-OH of 7.1 kJAEmol )1 and asparagine at position 39 disrupted a favorable interaction with this same hydroxyl. In conclusion, it is proposed that the specificity of new b-glycosidase mutants can be predicted by combining and adding energy of the enzyme-substrate interactions evaluated in the present study.Keywords: b-glycosidase; bioenergetics analysis; enzyme specificity; glycoside hydrolase; site-directed mutagenesis.b-glycosidases from glycoside hydrolase family 1 are enzymes that remove monosaccharides from the nonreducing end of di-and/or oligosaccharides. The nonreducing monosaccharide residue binds at the glycone subsite (subsite )1), whereas the remaining part of the substrate is accommodated by the aglycone subsite 2 , which may actually be composed of several subsites (+1, +2, +3, etc.). According to the CAZy database, family 1 currently comprises 427 b-glycosidases, with 3D structural data being available for 12 [1]. All family 1 b-glycosidases share the same tertiary structure [(b/a) 8 barrel]; they are configuration-retaining glycosidases, the catalytic activity of which depends on two glutamic acid residues, one positioned after the b strand 4 (catalytic proton donor) and the other after the b strand 7 (catalytic nucleophile). Family 1 comprises enzymes with 14 different EC numbers 3 , catalyzing hydrolysis of substrates presenting a variety of glycones (monosaccharides such as glucose, galactose, fucose, mannose, 6-phosphoglucose and 6-phosphogalactose) and aglycones (monosaccharides, oligosaccharides, alkyl and aryl moieties) [1]. This broad substrate specificity makes family 1 an interesting model for using to study the molecular basis of the enzymatic specificity.Having a better understanding of the molecular basis of b-glycosidase specificity wo...

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

“…The double mutations modify k cat 10 2 -to 10 3 -fold, whereas K m presents smaller changes. This pattern was also observed for the single mutations of residues Q39 and E451 (12,13). Thermal inactivation experiments showed that the structures of the A451E39 and S451E39 double mutants are similar to the wild-type Sfβgly (t ½ ~1.2 min at 50°C), suggesting that the mutational effects on k cat did not result from large structural modifications of the protein.…”

“…These single mutations caused a large reduction in the k cat /K m ratio for the hydrolysis of NPβglycosides due to a reduction of about 70% in the energy of the interactions formed by Q39 and E451 with the substrate in the ES ‡ complex (13). Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in BL21 DE3 cells in order to determine interactions between sites of mutation.…”

“…Single mutations of these residues showed that replacement of Q39 with E and N (glutamate and asparagines) causes a drastic decrease (about 70%) in the energy of the interactions with the glycone hydroxyls 4 and 6. In addition, substitution of E451 with Q did not affect the interactions with glycone hydroxyl 4, whereas substitutions with D and S (aspartate and serine) produced large reductions in the energy of these interactions (13). However, these interactions were analyzed on a single site mutant so that the effect of mutation of one residue on the interactions formed by a second was not studied.…”

Using the Amino Acid Network to Modulate the Hydrolytic Activity of β-Glycosidases

“…Kinetics from mutations D84A, R97A, S247A, N249A, F251A, F334A, L350A, K366A and Y420A are new data presented here. Remaining kinetic data collected from 1: Mendonça and Marana, 2011 [9]; 2: Marana et al ., 2002 [7]; 3: Marana et al ., 2004 [8]; 4: Tamaki et al ., 2014 [14]; 5: Mendonça and Marana, 2008 [11]; 6: Marana et al ., 2003 [6]. For calculation of Relative k cat / K m , each k cat / K m mut was compared to the k cat / K mWT data presented on the same manuscript in which the mutant enzyme was firstly described: 1: Mendonça and Marana, 2011 [9]; 2: Marana et al ., 2002 [7]; 3: Marana et al ., 2004 [8]; 4: Tamaki et al ., 2014 [14]; 5: Mendonça and Marana, 2008 [11]; 6: Marana et al ., 2003 [6] (*: This mutant was studied using MUβglc as substrate).…”

Methods for discovery and characterization of cellulolytic enzymes from insects

“…Methods based on HPLC or thin layer chromatography separations to estimate produced sugars have also been described (Adams & Drew, 1965; Berlin et al , 2006; Chundawat et al , 2008). To further characterize the activity of specific cellulolytic enzymes, activity is measured by spectrophotometry using substrates containing p ‐nitrophenol (Terra et al , 1979; Chipoulet & Chararas, 1985a; Cazemier et al , 1997; Marana et al , 2000; Ferreira et al , 2001; Marana et al , 2004; Yapi et al , 2009) or methyl‐umbelliferyl (MU) groups (Jacobson & Schlein, 1997; Marana et al , 2001).…”