Crystal Structure of Alkalophilic Asparagine 233-Replaced Cyclodextrin Glucanotransferase Complexed with an Inhibitor, Acarbose, at 2.0 Å Resolution

抄録

The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces a-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly α-1, β-, and γ-cyclodextrins, catalyzing the conversion of starch into cyclic or linear α-1, 4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 Å resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like α-cyclodextrin.