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Structural and mutational analysis of substrate recognition in kojibiose phosphorylase
Author(s) -
Okada Satoshi,
Yamamoto Takuo,
Watanabe Hikaru,
Nishimoto Tomoyuki,
Chaen Hiroto,
Fukuda Shigeharu,
Wakagi Takayoshi,
Fushinobu Shinya
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12622
Subject(s) - glycoside hydrolase , biochemistry , chemistry , glycogen phosphorylase , phosphorolysis , stereochemistry , enzyme , purine nucleoside phosphorylase , purine
Glycoside hydrolase ( GH ) family 65 contains phosphorylases acting on maltose ( G lc‐α1,4‐ G lc), kojibiose ( G lc‐α1,2‐ G lc), trehalose ( G lc‐α1,α1,‐ G lc), and nigerose ( G lc‐α1,3‐ G lc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α‐glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from C aldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, T rp391, G lu392, and T hr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that T rp391 and G lu392, especially the latter, are required for the kojibiose activity. Database Structural data are available in the Protein Data Bank database under accession numbers 3WIQ and 3WIR . Structured digital abstractCsKP and CsKP bind by x-ray crystallography ( View interaction ) .