Application | Comment | Organism |
---|---|---|
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 (Comment) | Organism |
---|---|
recombinant expression of wild-type and mutant enzymes | Caldicellulosiruptor saccharolyticus |
Crystallization (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
E392R | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
E392R/T417A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
E392R/T417I | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
T417A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
T417F | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
T417I | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
W391M/E392V | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
W391M/E392V/T417A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
W391M/E392V/T417I | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-alpha-D-glucosyl-D-glucose + phosphate | Caldicellulosiruptor saccharolyticus | - |
D-glucose + beta-D-glucose 1-phosphate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Caldicellulosiruptor saccharolyticus | A4XGP2 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes | Caldicellulosiruptor saccharolyticus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-alpha-D-glucosyl-D-glucose + phosphate | - |
Caldicellulosiruptor saccharolyticus | D-glucose + beta-D-glucose 1-phosphate | - |
r | |
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 | - |
? | |
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 | - |
? | |
maltose + phosphate | very low, activity, reaction of maltose phosphorylase, EC 2.4.1.8 | Caldicellulosiruptor saccharolyticus | D-glucose + beta-D-glucose 1-phosphate | - |
? | |
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 | Comment | Organism |
---|---|---|
CsKP | - |
Caldicellulosiruptor saccharolyticus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
assay at | Caldicellulosiruptor saccharolyticus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
assay at | Caldicellulosiruptor saccharolyticus |
General Information | Comment | Organism |
---|---|---|
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 |
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 |