Generation of amylosucrase variants that terminate catalysis of acceptor elongation at the di- or trisaccharide stage.
An amylosucrase gene was subjected to high-rate segmental random mutagenesis, which was directed toward a segment encoding amino acids that influence the interaction with substrate molecules in subsites -1 to +3. A screen was used to identify enzyme variants with compromised glucan chain elongation. With an average mutation rate of about one mutation per targeted codon, a considerable fraction (82%) of the clones that retained catalytic activity were deficient in this trait. A detailed characterization of selected variants revealed that elongation terminated when chains reached lengths of only two or three glucose moieties. Sequencing showed that the amylosucrase derivatives had an average of no more than two amino acid substitutions and suggested that predominantly exchanges of Asp394 or Gly396 were crucial for the novel properties. Structural models of the variants indicated that steric interference between the amino acids introduced at these sites and the growing oligosaccharide chain are mainly responsible for the limitation of glucosyl transfers. The variants generated may serve as biocatalysts for limited addition of glucose moieties to acceptor molecules, using sucrose as a readily available donor substrate.
Department of Chemical Biology, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
Jens
J
Schneider
Christin
C
Fricke
Heike
H
Overwin
Birgit
B
Hofmann
Bernd
B
Hofer
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D004186:Disaccharidases / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009343:Neisseria / Q000201:enzymology; D017434:Protein Structure, Tertiary; D014312:Trisaccharides / Q000378:metabolism*
D001426:Bacterial Proteins; D050505:Mutant Proteins; D014312:Trisaccharides; D005964:Glucosyltransferases; C019700:amylosucrase; D004186:Disaccharidases