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Title
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Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011.
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Authors
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K.Haga,
R.Kanai,
O.Sakamoto,
M.Aoyagi,
K.Harata,
K.Yamane.
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Ref.
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J Biochem (tokyo), 2003,
134,
881-891.
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PubMed id
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Abstract
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The stacking interaction between a tyrosine residue and the sugar ring at the
catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13
enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase
(CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all
CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259
onto the sugar ring at subsite +2 is essentially conserved among CGTases.
F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor
binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33,
9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking
interaction at subsites -1 and +2 on the substrate binding of CGTases, we
analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A
resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the
inhibitor, acarbose. The refined structures revealed that acarbose molecules
bound to the Y100L mutant moved from the active center toward the side chain of
Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose
and subsites significantly diminished. The position of pseudo-tetrasaccharide
binding in the F183L/F259L mutant was closer to the non-reducing end, and the
torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and
subsites -2 to -1 on molecule 2 significantly changed compared with that of each
molecule of wild-type-acarbose complex to adopt the structural change of subsite
+2. These structural and biochemical data suggest that substrate binding in the
active site of CGTase is critically affected by the carbohydrate/aromatic
stacking interaction with Tyr100 at the catalytic subsite -1 and that this
effect is likely a result of cooperation between Tyr100 and Phe259 through
stacking interaction with substrate at subsite +2.
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