3LIH

Crystal structure of fructosyltransferase (D191A) from A. japonicus in complex with raffinose

Summary for 3LIH

• Entry DOI: 10.2210/pdb3lih/pdb
• Related: 3LDK 3LDR 3LEM 3LF7 3LFI 3LIG
• Related PRD ID: PRD_900002
• Descriptor: Fructosyltransferase, alpha-D-galactopyranose-(1-6)-alpha-D-glucopyranose-(1-2)-beta-D-fructofuranose (3 entities in total)
• Functional Keywords: protein-oligosaccharide complex, five bladed beta-propeller fold, hydrolase
• Biological source: Aspergillus japonicus
• Total number of polymer chains: 1
• Total formula weight: 69447.00

Authors

Chuankhayan, P.,Chen, C.J.,Chiang, C.M. (deposition date: 2010-01-24, release date: 2010-05-12, Last modification date: 2024-03-20)

Primary citation

Chuankhayan, P.,Hsieh, C.Y.,Huang, Y.C.,Hsieh, Y.Y.,Guan, H.H.,Hsieh, Y.C.,Tien, Y.C.,Chen, C.D.,Chiang, C.M.,Chen, C.J.
Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis
J.Biol.Chem., 285:23251-23264, 2010

PubMed Abstract: 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.

PubMed: 20466731
DOI: 10.1074/jbc.M110.113027

Experimental method

X-RAY DIFFRACTION (2.2 Å)