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J Biol Chem, Vol. 273, Issue 10, 5771-5779, March 6, 1998

Engineering of Cyclodextrin Product Specificity and pH Optima of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

Richèle D. Wind, Joost C. M. Uitdehaag^¶, Reinetta M. Buitelaar, Bauke W. Dijkstra^¶, and Lubbert Dijkhuizen

From the  Agrotechnological Research Institute (ATO-DLO), P. O. Box 17, 6700 AA Wageningen, ^¶ BIOSON Research Institute and Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9747 AG Groningen, and  Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 -CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Å resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced -cyclodextrin production of the CGTase.

The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (- and -CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of -CD.

Glu²⁵⁸ is involved in catalysis in CGTases as well as -amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu²⁵⁸ in the CGTase from T. thermosulfurigenes EM1 were changed. Phe²⁸⁴ was replaced by Lys and Asn³²⁷ by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu²⁵⁸.

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