Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis.
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.
Chuankhayan
Phimonphan
P
Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan.
Hsieh
Chih-Yu
CY
Huang
Yen-Chieh
YC
Hsieh
Yi-You
YY
Guan
Hong-Hsiang
HH
Hsieh
Yin-Cheng
YC
Tien
Yueh-Chu
YC
Chen
Chung-De
CD
Chiang
Chien-Min
CM
Chen
Chun-Jung
CJ
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D001426:Bacterial Proteins
D004791:Enzyme Inhibitors; D005947:Glucose; D006026:Glycoside Hydrolases; D006602:Hexosyltransferases; C021451:inulosucrase; D001426:Bacterial Proteins
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