Structural basis of a mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from...

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,b). In this study, we determined one X-ray structure of the mutant Y195I α-CGTase at 2.3Å. 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.