Structural and functional studies of inulin fructotransferase and phytotoxin toxoflavin-degrading enzyme

Abstract

Inulin fructotransferase (IFTase), a member of glycoside hydrolase family 91, catalyzes a depolymerization of β-2,1-fructans inulin by successively removing the terminal difructosaccharide units as cyclic anhydrides via intramolecular fructosyl transfer. The crystal structures of IFTase and its substrate-bound complex reveal that IFTase is a trimeric enzyme, and each monomer folds into a right-handed parallel β-helix. Despite variation in the number and conformation of its β-strands, the IFTase β-helix has a structure that is largely reminiscent of other β-helix structures, but is unprecedented in that trimerization is a prerequisite for catalytic activity and the active site is located at the monomer-monomer interface. Results from crystallographic studies and site-directed mutagenesis provide a structural basis for the exolytic-type activity of IFTase, and a functional resemblance to inverting-type glycosyltransferases. Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors, which play essential roles in the pathogenicity of bacteria in various hosts. Therefore, these chemicals are targets for antivirulence strategies. The phytopathogen Burkholderia glumae BGR1 produces a phytotoxin, toxoflavin, which is the key factor in bacterial wilting of crop plants. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2. Here, the crystal structure of TxDE in the substrate-free form at 1.6 Å resolution and in complex with toxoflavin at 2.0 Å resolution is reported along with the results of a functional analysis. The overall structure of TxDE is similar to the structures of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. The binding of substrate did not cause any noticeable conformational changes in the enzyme, and toxoflavin binds in the Mn(II)-coordination shell by replacing ligating water molecules. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol (DTT). These results provide insight into the catalytic mechanism of TxDE, its recently proposed role as a non-antibiotic selection marker for plants, and its function as an antivirulence factor in toxoflavin-mediated plant diseases.