Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

HERNAN COSTA, SERGIO DEL CANTO, SUSANA FERRAROTTI, MIRTHA BISCOGLIO DE JIMENEZ BONINO

CARBOHYDRATE RESEARCH

ELSEVIER

Año: 2009 vol. 344 p. 74 - 79

0008-6215

Cyclodextrin Glycosyltransferases catalyse cyclodextrin production from starch and other related carbohydrates, an important industrial process. Several studies of structure-function relationship have been conducted with ethoxyformyc anhydride as a selective modifier of histidine residues and three histidines were considered essential for enzyme biological properties. We herein report the ethoxyformylation of the cyclodextrin glycosyltransferase from Bacillus circulans DF 9R with ethoxyformyc anhydride by using a reagent/protein ratio low enough to modified only one out of the 15 histidine residues of the enzyme this leading to a drastic drop in its cyclizing and hydrolyzing activity. Enzyme treatment with 14C ethoxyformyc anhydride, tryptic digestion, mass spectrometry and sequence analysis of the peptide mixture allowed us to show that histidine 233 is the most reactive histidine residue, to find that this enzyme is the only member of the 39 CGTase group having glutamine instead of glycine in the -6 subsite, a very conserved region included in the substrate binding cleft and to contribute to a further enzyme characterization. These data will be useful to obtain the recombinant protein as well as to perform protein engineering experiments leading to industrial applications.