Structural basis of a mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to β-/γ-cyclodextrin

Cyclodextrin glycosyltransferase (EC 2.4.1.19) (CGTase) is an extracellular bacterial enzyme which has the unique capability of forming cyclodextrins from starch. Our previous investigation revealed that a mutant Y195I α-CGTase drastically altered the cyclodextrin specificity by switching toward the synthesis of both β- and γ-CDs (Xie et al., 2013a, Xie et al., 2013b). In this study, we determined one X-ray structure of the mutant Y195I α-CGTase at 2.3 Å. The overall structure was similar to that of the typical β-CGTase from Bacillus circulans 251, with minor difference in flexible domains since they showed about 70% homogeneity of amino acid sequences. The central site with isoleucine tended to be more flexible than tyrosine thus made the sugar chain, during the cyclization process, form a larger cyclodextrin like β- and γ-CDs surrounding the central site instead of α-CD. Superposition of the structure of Y195I α-CGTase with those of β-CGTase and γ-CGTase showed that residues Lys232, Lys89 and Arg177 at subsites +2, −3 and −7 could form smaller substrate binding cavity. In summary, the crystal structure revealed that moderate increase of mobility of the central site resulted in the switched product specificity from α-CD to β- and γ-CDs of the mutant Y195I α-CGTase. The space differences alongside the active domain may be another factor that impacts the product specificity of the CGTase.