Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity.
Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.
Leemhuis
Hans
H
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Kragh
Karsten M
KM
Dijkstra
Bauke W
BW
Dijkhuizen
Lubbert
L
D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics*; D006867:Hydrolases / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D013213:Starch / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry*
D011994:Recombinant Proteins; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D006867:Hydrolases; D000516:alpha-Amylases