Site-directed mutagenesis establishes aspartic acids-227 and -342 as essential for enzyme activity in an isomalto-dextranase from Arthrobacter globiformis.
Isomalto-dextranase, from Arthrobacter globiformis T6, is a member of the glycoside hydrolase family 27. However, the alignments of the whole amino acid sequence are distinct from other members of this family. The enzymes cleave the glycosidic bond of the substrate in two different manners: either retaining or inverting the anomeric configuration. We believe that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomalto-dextranase active site that catalyzes the hydrolysis reaction of dextran have been identified and the roles of nine amino acid residues (D107, D163, D227, D295, D340, D342, D373, D396, and E420) in the isomalto-dextranase from A. globiformis analyzed by site-directed mutagenesis. Of 15 mutant enzymes that were prepared, eight had reduced activities for dextran hydrolysis. Aspartic acids-227 and -342, which are part of the apparent catalytic dyad, were essential for hydrolase activity toward dextran.
Tochihara
Takashi
T
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Sasaki
Katsutomo
K
Araki
Osamu
O
Morimoto
Naoki
N
Watanabe
Kenji
K
Hatada
Yuji
Y
Ito
Susumu
S
Ito
Hiroyuki
H
Matsui
Hirokazu
H
D000818:Animals; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment
D011994:Recombinant Proteins; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; C091051:glucan 1,6-alpha-isomaltosidase