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Protein Science (2004), 13:457-465. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site

Ryuta Kanai1,2, Keiko Haga1, Toshihiko Akiba2, Kunio Yamane1 and Kazuaki Harata2

1 Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
2 Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan

(RECEIVED September 5, 2003; FINAL REVISION October 29, 2003; ACCEPTED October 31, 2003)



Abstract

Cyclodextrin glycosyltransferase (CGTase) belonging to the {alpha}-amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related {alpha}-1,4-glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in {alpha}-amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose-complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y). The temperature factors of the region 259–269 in native F283L increased >10 Å2 compared with the wild type. The complex formation with acarbose not only increased the temperature factors (>10 Å2) but also changed the structure of the region 257–267. This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding. The conformation of the side-chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259. The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range. The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pKa of Glu257 in F283Y may be lower than that in the wild type.

Keywords: CGTase; crystal structure; substrate binding; catalytic activity

Reprint requests to: Kazuaki Harata, Biological Information Research Center, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; e-mail: k-harata{at}aist.go.jp; fax: 81-29-861-6194.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03408504.


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R. M. Kelly, H. Leemhuis, L. Gatjen, and L. Dijkhuizen
Evolution toward Small Molecule Inhibitor Resistance Affects Native Enzyme Function and Stability, Generating Acarbose-insensitive Cyclodextrin Glucanotransferase Variants
J. Biol. Chem., April 18, 2008; 283(16): 10727 - 10734.
[Abstract] [Full Text] [PDF]


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