The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase

Abstract

Cyclodextrin glycosyltransferases (CGTases)are important enzymes in biotechnology because oftheir ability to produce cyclodextrin (CD) mixtures fromstarch whose relative composition depends on enzymesource. A multiple alignment of 46 CGTases andShannon entropy analysis allowed us to find differencesand similarities that could be related to productspecificity. Interestingly, position 179 has Gly in allthe CGTases except in that from Bacillus circulans DF9R which possesses Gln. The absence of a side chain atthat position has been considered as a strong requirementfor substrate binding and cyclization process. Therefore,we constructed two mutants of this enzyme, Q179L andQ179G. The activity and kinetic parameters of Q179Gremained unchanged while the Q179L mutant showed adifferent CDs ratio, a lower catalytic efficiency, and adecreased ability to convert starch into CDs. We show thatposition 179 is involved in CGTase product specificity andmust be occupied by Gly?without a side chain?or byamino acid residues able to interact with the substratethrough hydrogen bonds in a way that the cyclizationprocess occurs efficiently. These findings are alsoexplained on the basis of a structural model.