title: Enhanced catalytic efficiency in quercetin-4'-glucoside hydrolysis of Thermotoga maritima β-glucosidase A by site-directed mutagenesis.
abstract: Te-BglA and Tm-BglA are glycoside hydrolase family 1 β-glucosidases from Thermoanaerobacter ethanolicus JW200 and Thermotoga maritima, respectively, with 53% sequence identity. However, Te-BglA could more effectively hydrolyze isoflavone glucosides to their aglycones than could Tm-BglA, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of Tm-BglA with the corresponding residues of Te-BglA, generating three single mutants (F221L, N223L, and G224T), as well as the corresponding three double mutants (F221L/N223L, F221L/G224T, and N223L/G224T) and one triple mutant (F221L/N223L/G224T). The seven mutants have been purified, characterized, and compared to the wild-type Tm-BglA. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. All mutants showed pH-activity curves narrower on the basic side and wider on the acid side and had similar optimal pH and stability at pH 6.5-8.3. They were more stable up to 85 °C, but G224T displayed higher optimal temperature than Tm-BglA. Seven mutants indicated an obvious increase in catalytic efficiency toward p-nitrophenyl β-D-glucopyranoside (pNPG) but an increase or not change in K(m). All mutants showed a decrease in catalytic efficiency of isoflavonoid glycosides and were not changed for F221L and lost for N223L in enzymatic hydrolysis on quercetin glucosides. Contrarily, G224T resulted in a dramatic increase conversion of Q4' (35.5%) and Q3,4' (28.6%) in accord with an increased turnover number (k(cat), 1.4×) and catalytic efficiency (k(cat)/K(m), 2.2×) as well as a decrease in K(m) (0.24) for Q4'. Modeling showed that G224T mutation at position 224 may enhance the interaction between G224T and 5-OH and 3-OH on the quercetin backbone of Q4'.
authors.affiliation: Department of Food Science and Nutrition, GinLing College, Nanjing Normal University , Nanjing, People's Republic of China 210097.
authors.forename: Huihui
authors.initials: H
authors.lastname: Sun
authors.forename: Yemin
authors.initials: Y
authors.lastname: Xue
authors.forename: Yufei
authors.initials: Y
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007529:Isoflavones / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D011794:Quercetin / Q000031:analogs & derivatives* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D007529:Isoflavones; D011794:Quercetin; D001617:beta-Glucosidase; C080613:spiraeoside