ENGINEERING OF CYCLODEXTRIN PRODUCT SPECIFICITY AND PH OPTIMA OF THE THERMOSTABLE CYCLODEXTRIN GLYCOSYLTRANSFERASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1

The product specificity and pH optimum of the thermostable cyclodextri n glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfur igenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experime nt with the Bacillus circulans strain 251 beta-CGTase had revealed a m altohexaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM 1 resulted in a 2.6-Angstrom resolution x-ray structure of a complex w ith a maltohexaose inhibitor, bound in a different conformation, We hy pothesize that the new maltohexaose conformation is related to the enh anced alpha-cyclodextrin production of the CGTase. The detailed struct ural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by si te directed mutagenesis, Mutation D371R was aimed at hindering the mal tohexaose conformation and resulted in enhanced production of larger s ize cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at st abilization of the new maltohexaose conformation and resulted in incre ased production of alpha-CD. Glu(258) is involved in catalysis in CGTa ses as well as alpha-amylases, and is the proton donor in the first st ep of the cyclization reaction. Amino acids close to Glu(258) in the C GTase from T. thermosulfurigenes EM1 were changed. Phe(284) was replac ed by Lys and Asn(327) by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrol ysis when compared with the wild-type enzyme, This suggests that the p H optimum curve of CGTase is determined only by residue Glu(258).