Crystal structure of a glycoside hydrolase family 68 β-fructosyltransferase from <i>Beijerinckia indica</i> subsp. <i>indica</i> in complex with fructose

Abstract

An enzyme belonging to glycoside hydrolase family 68 (GH68) from <i>Beijerinckia indica</i> subsp. <i>indica</i> NBRC 3744 was expressed in <i>Escherichia coli</i>. Biochemical characterization showed that the enzyme was identified to be a β-fructosyltransferase (BiBftA). Crystallization of a full-length BiBftA was initially attempted, but no crystals were obtained. We constructed a variant in which 5 residues (Pro199-Gly203) and 13 residues (Leu522-Gln534) in potentially flexible regions were deleted, and we successfully crystallized this variant BiBftA. BiBftA is composed of a five-bladed β-propeller fold as in other GH68 enzymes. The structure of BiBftA in complex with fructose unexpectedly indicated that one β-fructofuranose (β-Fru<i>f</i>) molecule and one β-fructopyranose molecule bind to the catalytic pocket. The orientation of β-Fru<i>f</i> at subsite -1 is tilted from the orientation observed in most GH68 enzymes, presenting a second structure of a GH68 enzyme in complex with the tilted binding mode of β-Fru<i>f.</i>