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Literature summary extracted from

  • Okada, S.; Yamamoto, T.; Watanabe, H.; Nishimoto, T.; Chaen, H.; Fukuda, S.; Wakagi, T.; Fushinobu, S.
    Structural and mutational analysis of substrate recognition in kojibiose phosphorylase (2014), FEBS J., 281, 778-786.
    View publication on PubMed

Application

EC Number Application Comment Organism
2.4.1.230 synthesis the phosphorylase can efficiently catalyze the reverse reaction with high specificity, and thus can be applied to the practical synthesis of alpha-glucosyl oligosaccharides Caldicellulosiruptor saccharolyticus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.230 recombinant expression of wild-type and mutant enzymes Caldicellulosiruptor saccharolyticus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.230 purified enzyme in complex with glucose and phosphate and in complex with kojibiose and sulfate, Glc-PO4 crystals are obtained at 25°C by sitting drop vapor diffusion method, mixing 0.001 ml of 5.9 m/ml protein in 5 mM Tris/HCl, pH 7.5, with an equal volume of reservoir solution containing 5 mM D-glucose, 5 mM sodium phosphate, pH 8.5, 10% v/v 2-propanol, 10% w/v PEG 3350, and 0.1 M Tris/HCl, pH 8.5, the kojibiose-SO4 crystals are obtained at 25°C by hanging drop vapor diffusion method, mixing of 0.001 ml of 5.4 mg/ml protein solution in 5 mM Tris/HCl, pH 7.5, with an equal volume of reservoir solution containing 5 mM kojibiose, 0.2 M NaCl, 2.0 M (NH4)2SO4, and 0.1 M sodium cacodylate, pH 6.5, X-ray diffraction structure determination and analysis at 2.05-2.80 A resolution, structures PDB IDs 3WIR and 3WIQ, molecular replacement with the maltose phosphorylase structure from Lactobacillus brevis LbMP, PDB ID 1H54, as the search model Caldicellulosiruptor saccharolyticus

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.230 E392R site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 E392R/T417A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 E392R/T417I site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 T417A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 T417F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 T417I site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 W391M/E392V site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 W391M/E392V/T417A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus
2.4.1.230 W391M/E392V/T417I site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Caldicellulosiruptor saccharolyticus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.1.230 2-alpha-D-glucosyl-D-glucose + phosphate Caldicellulosiruptor saccharolyticus
-
 D-glucose + beta-D-glucose 1-phosphate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.230 Caldicellulosiruptor saccharolyticus A4XGP2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.230 recombinant wild-type and mutant enzymes Caldicellulosiruptor saccharolyticus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.230 2-alpha-D-glucosyl-D-glucose + phosphate
-
 Caldicellulosiruptor saccharolyticus D-glucose + beta-D-glucose 1-phosphate
-
 r
2.4.1.230 3-O-alpha-D-glucopyranosyl-D-glucopyranose + phosphate low activity, reaction of nigerose phosphorylase, EC 2.4.1.279 Caldicellulosiruptor saccharolyticus D-glucose + beta-D-glucose 1-phosphate
-
 ?
2.4.1.230 alpha,alpha-trehalose + phosphate very low activity, reaction of trehalose phosphorylase, EC 2.4.1.64 Caldicellulosiruptor saccharolyticus D-glucose + beta-D-glucose 1-phosphate
-
 ?
2.4.1.230 maltose + phosphate very low, activity, reaction of maltose phosphorylase, EC 2.4.1.8 Caldicellulosiruptor saccharolyticus D-glucose + beta-D-glucose 1-phosphate
-
 ?
2.4.1.230 additional information nigerose, maltose, and trehalose are minor substrates for the wild-type enzyme, substrate specificity of wild-type and mutant enzymes, overview Caldicellulosiruptor saccharolyticus ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.4.1.230 CsKP
-
 Caldicellulosiruptor saccharolyticus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.230 50
-
 assay at Caldicellulosiruptor saccharolyticus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.230 6
-
 assay at Caldicellulosiruptor saccharolyticus

General Information

EC Number General Information Comment Organism
2.4.1.230 evolution the kojibiose phosphorylase belongs to the glycoside hydrolase (GH) family 65, that contains phosphorylases acting on maltose (Glc-alpha1,4-Glc), kojibiose (Glc-alpha1,2-Glc), trehalose (Glc-alpha1,alpha1,-Glc), and nigerose (Glc-alpha1,3-Glc). 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 Caldicellulosiruptor saccharolyticus
2.4.1.230 additional information the loop 3 region comprises the active site of kojibiose, three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Trp391 and Glu392, especially the latter, are required for the kojibiose activity, active site structure and substrate binding, overview. Comparison of substrate recognition by kojibiose phosphorylase CsKP from Caldicellulosiruptor saccharolyticus and maltose phosphorylase LbMP from Lactobacillus brevis, modeling, overview Caldicellulosiruptor saccharolyticus