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J. Biol. Chem., Vol. 277, Issue 2, 1113-1119, January 11, 2002

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The Remote Substrate Binding Subsite 6 in Cyclodextrin-glycosyltransferase Controls the Transferase Activity of the Enzyme via an Induced-fit Mechanism^*

Hans Leemhuis, Joost C. M. Uitdehaag^§, Henriëtte J. Rozeboom^§, Bauke W. Dijkstra^§, and Lubbert Dijkhuizen^¶

From the  Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren and the ^§ BIOSON Research Institute and Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of -, -, and -cyclodextrins (cyclic -(1,4)-linked oligosaccharides of 6, 7, or 8 glucose residues, respectively) from starch. Nine substrate binding subsites were observed in an x-ray structure of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite 6 is conserved in CGTases, suggesting its importance for the reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G, N193L, and G179L/G180L). All subsite 6 mutants had decreased k_cat values for -cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis. Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions. The results demonstrate that (i) subsite 6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii) Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently change cyclization and coupling activities, and (iv) substrate interactions at subsite 6 activate the enzyme in catalysis via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit mechanism in the -amylase family.

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