Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production

Cyclodextrin glycosyltransferases (CGTase) (EC;2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTase s produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans st rain 251 revealed sugar binding subsites, distant from the catalytic residu es, which have been proposed to be involved in the cyclodextrin size specif icity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mu tant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. Th e mutations affected the cyclization, coupling; disproportionation and hydr olyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twof old increase in the production of alpha-cyclodextrin from starch. This muta nt protein was crystallized and its X-ray structure, in a complex with a ma ltohexaose inhibitor, was determined at 2.4 Angstrom resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose i nhibitor, allowing rationalization of the observed change in product specif icity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contr ibute strongly to the size of cyclodextrin products formed and thus to CGTa se product specificity. Changes in sugar binding subsites -3 and -7 thus re sult in mutant proteins with changed cyclodextrin production specificity. ( C) 2000 Academic Press.