Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 A resolution.
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 alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-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 A 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 a-cyclodextrin.
Ishii
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N
Biophysical Chemistry Laboratory, National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki 305-8566, Japan. ishii@nibh.go.jp.
Haga
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K
Yamane
K
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Harata
K
K
D020909:Acarbose / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005964:Glucosyltransferases / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary
D001216:Asparagine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D020909:Acarbose