The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 implications for product inhibition and product specificity.

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active-site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products. Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins. Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47-->Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47-->Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity.