title: Structural and mutational analysis of substrate recognition in kojibiose phosphorylase.
abstract: Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-α1,4-Glc), kojibiose (Glc-α1,2-Glc), trehalose (Glc-α1,α1,-Glc), and nigerose (Glc-α1,3-Glc). 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 Caldicellulosiruptor 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, Trp391, Glu392, and Thr417, 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 Trp391 and Glu392, especially the latter, are required for the kojibiose activity.
authors.affiliation: Department of Biotechnology, University of Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okada
authors.forename: Takuo
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Hikaru
authors.initials: H
authors.lastname: Watanabe
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nishimoto
authors.forename: Hiroto
authors.initials: H
authors.lastname: Chaen
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Fukuda
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Wakagi
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry / Q000378:metabolism; D046968:Thermoanaerobacter / Q000201:enzymology*; D013912:Threonine / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D050505:Mutant Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D013431:Sulfates; D013912:Threonine; D018698:Glutamic Acid; D014364:Tryptophan; C008237:kojibiose; D005964:Glucosyltransferases; D006026:Glycoside Hydrolases; D005947:Glucose
keywords: enzyme-substrate interactions; maltose phosphorylase; oligosaccharide synthesis; structure-function relationships; substrate specificity