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

• Published in: Bioscience, biotechnology, and biochemistry Vol. 84; no. 12; pp. 2508 - 2520
• Main Authors: Tonozuka, Takashi, Kitamura, Junichi, Nagaya, Mika, Kawai, Reika, Nishikawa, Atsushi, Hirano, Katsuaki, Tamura, Keisuke, Fujii, Tadashi, Tochio, Takumi
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 01.12.2020; Oxford University Press
• Subjects: Amino Acid Sequence; Beijerinckia indica; Beijerinckiaceae - enzymology; Crystallography, X-Ray; fructooligosaccharide; Fructose - metabolism; GH68; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Models, Molecular; Mutagenesis; Protein Conformation; protein crystallization; Structure-Activity Relationship; β-fructosyltransferase; fructooligosaccharide; GH68; β-fructosyltransferase; protein crystallization; Beijerinckia indica
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1080/09168451.2020.1804317

An enzyme belonging to glycoside hydrolase family 68 (GH68) from Beijerinckia indica subsp. indica NBRC 3744 was expressed in Escherichia coli. 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-Fruf) molecule and one β-fructopyranose molecule bind to the catalytic pocket. The orientation of β-Fruf 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-Fruf. Structure of the GH68 enzyme unexpectedly indicated that one β-fructofuranose and one β-fructopyranose bind to the catalytic pocket.