Abstract
The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-A structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and +1, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.
Highlights
Function in CGTasedThe architecture of subsite Ϫ1 is conserved in the entire ␣-amylase family [3, 7]. Ser/Met/Leu/
The enzyme cyclodextrin glycosyltransferase is closely related to ␣-amylases but has the unique ability to produce cyclodextrins (circular ␣(134)-linked glucoses) from starch
The active site of cyclodextrin glycosyltransferase (CGTase) is an interesting example of an architecture in which a particular specificity is added to a conserved ␣-amylase bond cleavage machinery
Summary
The architecture of subsite Ϫ1 is conserved in the entire ␣-amylase family [3, 7]. Ser/Met/Leu/. Donor Cyclization and specificity [44, 49] Cyclization and specificity [49] Cyclizatione [17] Cyclizatione [17] Cyclizatione
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