Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity

The enzymes from the cl-amylase family all share a similar alpha-retaining catalytic mechanism but can have different reaction and product specificiti es. One family member, cyclodextrin glycosyltransferase (CGTase), has an un commonly high transglycosylation activity and is able to form cyclodextrins . We have determined the 2.0 and 2.5 Angstrom X-ray structures of E257A/D22 9A CGTase in complex with maltoheptaose and maltohexaose. Both sugars are b ound at the donor subsites of the active site and the acceptor subsites are empty. These structures mimic a reaction stage in which a covalent enzyme- sugar intermediate awaits binding of an acceptor molecule. Comparison of th ese structures with CGTase-substrate and CGTase-product complexes reveals t hree different conformational states for the CGTase active site that are ch aracterized by different orientations of the centrally located residue Tyr 195. In the maltoheptaose and maltohexaose-complexed conformation, CGTase h inders binding of an acceptor sugar at subsite +1, which suggests an induce d-fit mechanism that could explain the transglycosylation activity of CGTas e. In addition, the maltoheptaose and maltohexaose complexes give insight i nto the cyclodextrin size specificity of CGTases, since they precede alpha- cyclodextrin (six glucoses) and beta-cyclodextrin (seven glucoses) formatio n, respectively. Both ligands show conformational differences at specific s ugar binding subsites, suggesting that these determine cyclodextrin product size specificity, which is confirmed by site-directed mutagenesis experime nts.