Mutations converting cyclodextrin glycosyltransferase from a transglycosylase into a starch hydrolase.
Cyclodextrin glycosyltransferase (CGTase) efficiently catalyzes transglycosylation of oligo-maltodextrins, although the enzyme also has a low hydrolytic activity. Its +2 substrate binding subsite, which contains the conserved Phe184 and Phe260 residues, has been shown to be important for this transglycosylation activity [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. Here we show that the amino acid side chain at position 260 also controls the hydrolytic activity of CGTase. Three Phe260 mutants of Thermoanaerobacterium thermosulfurigenes CGTase were obtained with a higher hydrolytic activity than ever observed before for a CGTase. These Phe260 mutations even changed CGTase from a transglycosylase into a starch hydrolase.
Leemhuis
Hans
H
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Dijkstra
Bauke W
BW
Dijkhuizen
Lubbert
L
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D003013:Clostridium; D003505:Cyclodextrins / Q000737:chemistry; D004789:Enzyme Activation / Q000502:physiology; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D003505:Cyclodextrins; D010649:Phenylalanine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D006026:Glycoside Hydrolases