Altering the substrate chain-length specificity of an alpha-glucosidase.
Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase.
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 07 Aza Aobayama Aramaki, Aoba-ku, Sendai-shi, Miyagi-ken 980-8579, Japan.
Akio
A
Noguchi
Toru
T
Nakayama
Hisashi
H
Hemmi
Tokuzo
T
Nishino
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008024:Ligands; D009844:Oligosaccharides; D000520:alpha-Glucosidases