The Cyclization Mechanism of Cyclodextrin Glycosyltransferase (CGTase) as Revealed by a γ-Cyclodextrin-CGTase Complex at 1.8-Å Resolution

Abstract

The enzyme cyclodextrin glycosyltransferase is closely related to ␣-amylases but has the unique ability to produce cyclodextrins (circular ␣(134)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-Å structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product ␥-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.

Complex Structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose and Cyclodextrins Demonstrate the Multiple Substrate Recognition Mechanism

“…Therefore, the resultant structure of the ␥-CD complex is possibly the previous state of the structure of ␣-CD complex, from which the rotation of the indole ring of Trp 356 about 2 could lead to a similar structure of ␣-CD complex. This is supported by the fact that the ␥-CD binding to cyclodextrin glycosyltransferase can change the conformation of the glucosidic bond at the hydrolyzing site like acarbose because of the eight glucose units making the cyclic structure loose (35).…”

Oligosaccharide Binding to Barley α-Amylase 1

“…The crystal structure of the double mutant E257Q/D229N of CGTase from Bacillus circulans (strain 251) in complex with ␥-cyclodextrins showed that Tyr-195 in the active site is essential for the circularization mechanism (57). Remarkably, the phenolic ring of this residue is very close to the center of the ␥-cyclodextrin, and the binding of the molecule induces an important shift (2.6 Å) of this key residue.…”

Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites –6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley α-Amylase 1

“…Substrate analogs are very rarely seen to fill the entire binding site in crystal structures, one example being a Bacillus licheniformis/Bacillus amyloliquefaciens ␣-amylase chimera accommodating at subsites Ϫ7 through ϩ3 a decasaccharide inhibitor derived by transglycosylation from the pseudotetrasaccharide acarbose (14). Related inhibitors cover the only five subsite long binding crevice in pancreatic ␣-amylase (8,9,20), and occupy part of the longer binding sites in microbial ␣-amylases (11,13,21) and in cyclodextrin glucosyltransferase (CGTase) (16,22,23). The structures validate modeled substrate complexes and subsite maps (8,12,24,25) by highlighting (i) aromatic stacking and hydrogen bonds between carbohydrate and protein (9,10,21,24,26,27), (ii) conformational features of the bound carbohydrate (8,21,28), (iii) conserved geometry of the catalytic site (10,14,15,21,22,29,30), and (iv) substrate binding motifs in ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel (15).…”