Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics.
Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed. Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate. Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.
Nakamura
A
A
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Haga
K
K
Yamane
K
K
D020909:Acarbose; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D003500:Cyclization; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D013213:Starch / Q000378:metabolism; D013329:Structure-Activity Relationship; D014312:Trisaccharides / Q000494:pharmacology; D014443:Tyrosine
D014312:Trisaccharides; D014443:Tyrosine; D010649:Phenylalanine; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D020909:Acarbose