Crystal Structures Of Aspergillus Japonicus Fructosyltransferase Complex With Donor/Acceptor Substrates Reveal Complete Subsites In The Active Site For Catalysis

doi:10.1074/jbc.M110.113027

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標題:	Crystal Structures Of Aspergillus Japonicus Fructosyltransferase Complex With Donor/Acceptor Substrates Reveal Complete Subsites In The Active Site For Catalysis
作者:	Phimonphan Chuankhayan Chih-Yu Hsieh Yen-Chieh Huang Yi-You Hsieh Hong-Hsiang Guan Yin-Cheng Hsieh Yueh-Chu Tien Chung-De Chen Chien-Min Chiang Chun-Jung Chen
貢獻者:	生物科技系
關鍵字:	Aspergillus Crystal Structure Hydrolases Protein Structure X-ray Crystallography Fructooligosaccharide Fructosyltransferase Glycoside Hydrolase Substrate-binding Subsite Transfrucotosylation
日期:	2010-07
上傳時間:	2012-03-30 15:17:48 (UTC+8)
摘要:	Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade beta-propeller fold linked to a C-terminal beta-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.
關聯:	J. Biol. Chem. 285(30)：p.23251-23264
顯示於類別:	[生物科技系(所)] 期刊論文

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