Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis

J Biol Chem. 2010 Jul 23;285(30):23251-64. doi: 10.1074/jbc.M110.113027. Epub 2010 May 13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspergillus / enzymology*
  • Biocatalysis*
  • Catalytic Domain*
  • Crystallography, X-Ray
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Glucose / metabolism
  • Glucose / pharmacology
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / metabolism
  • Hexosyltransferases / antagonists & inhibitors
  • Hexosyltransferases / chemistry*
  • Hexosyltransferases / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Structure-Activity Relationship

Substances

  • Enzyme Inhibitors
  • Hexosyltransferases
  • inulosucrase
  • Glycoside Hydrolases
  • Glucose

Associated data

  • PDB/3LDK
  • PDB/3LDR
  • PDB/3LEM
  • PDB/3LF7
  • PDB/3LFI
  • PDB/3LIG
  • PDB/3LIH