Structural elements responsible for the glucosidic linkage-selectivity of a glycoside hydrolase family 13 exo-glucosidase.
Glycoside hydrolase family 13 contains exo-glucosidases specific for α-(1→4)- and α-(1→6)-linkages including α-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The α-(1→6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to α-(1→4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher kcat/Km for maltose than wild-type, but its kcat/Km for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher kcat/Km for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the α-(1→6)-glucosidic linkage.
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan.
Wataru
W
Saburi
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan.
Hiroaki
H
Rachi-Otsuka
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan; Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima, Hiroshima 739-8528, Japan.
Hironori
H
Hondoh
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan.
Masayuki
M
Okuyama
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan.
Haruhide
H
Mori
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8689, Japan. Electronic address: kimura@abs.agr.hokudai.ac.jp.
Atsuo
A
Kimura
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics*; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013295:Streptococcus mutans / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007534:Isomaltose; D008320:Maltose; D006026:Glycoside Hydrolases
Dextran glucosidase; Glycoside hydrolase family 13; Oligo-1,6-glucosidase; Substrate specificity; α-Glucosidase