Activating Compound | Comment | Organism | Structure |
---|---|---|---|
glucose 1,6-bisphosphate | required for activation. The conformational change in Lys240 alters the affinity of the enzyme for the activator | Bacillus cereus |
Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Bacillus cereus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant detagged wild-type and mutant T85Q and T85E enzymes free or in complex with glucose 1,6-bisphosphate, hanging drop vapor diffusion method, for the free enzyme crystals: mixing of 10 mg/ml protein in 25mM Tris-HCl, pH 7.5, and 1 mM MnCl2, with reservoir solution containing 100 mM Bis-Tris, pH 5.5, 50 mM MnCl2, 17% PEG 3350 (wild-type) or 13% PEG 3350 (mutant T85Q), and 75 mM NH4CH3COO, for the complex crystals: mixing of 10 mg/ml protein in 5 mM glucose 1,6-bisphosphate, 25 mM Tris-HCl, pH 7.4, and 1 mM MnCl2 with a reservoir solution containing 100 mM Bis-Tris, pH 5.5, 50 mM MnCl2, 14% PEG 3350, and 50 mM NH4CH3COO, X-ray diffraction structure determination and analysis at 1.8-2.3 A resolution | Bacillus cereus |
Protein Variants | Comment | Organism |
---|---|---|
D156A | site-directed mutagenesis, the D156A variant displays a dramatically reduced level of phosphorylation of the active site Thr85 compared to the wild-type, and does not acquire phosphatase activity | Bacillus cereus |
K240A | site-directed mutagenesis, the K240A variant can be phosphorylated by the small molecule activator glucose 1,6-bisphosphate like the wild-type enzyme | Bacillus cereus |
T85E | site-directed mutagenesis, the T85E variant mimics the active site charge of the activated state of the enzyme, the affinity for activator glucose 1,6-bisphosphate is 4.5fold reduced compared to the wild-type enzyme | Bacillus cereus |
T85Q | site-directed mutagenesis, the T85Q variant mimics the active site charge of the unactivated state of the enzyme, the affinity for activator glucose 1,6-bisphosphate is only slightly reduced compared to the wild-type enzyme | Bacillus cereus |
General Stability | Organism |
---|---|
the phosphoenzyme is stable throughout purification and crystallization | Bacillus cereus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | a di-Mn2+ enzyme | Bacillus cereus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
alpha-D-ribose 1-phosphate | Bacillus cereus | - |
alpha-D-ribose 5-phosphate | - |
r | |
alpha-D-ribose 1-phosphate | Bacillus cereus DSM 31 | - |
alpha-D-ribose 5-phosphate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus cereus | Q818Z9 | - |
- |
Bacillus cereus DSM 31 | Q818Z9 | - |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
phosphoprotein | phosphorylation of the active site nucleophile, Thr85, activates the enzyme, structure comparison of activated phosphorylated and unactivated unphosphorylated enzymes, phosphorylation-dependent interdomain orientation, overview | Bacillus cereus |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, His-tag removal by thrombin cleavage, followed by gel filtration | Bacillus cereus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
alpha-D-ribose 1-phosphate | - |
Bacillus cereus | alpha-D-ribose 5-phosphate | - |
r | |
alpha-D-ribose 1-phosphate | - |
Bacillus cereus DSM 31 | alpha-D-ribose 5-phosphate | - |
r |
Subunits | Comment | Organism |
---|---|---|
More | the active site is located between two independently folded domains | Bacillus cereus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
20 | 22 | assay at | Bacillus cereus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Bacillus cereus |
General Information | Comment | Organism |
---|---|---|
additional information | the active site is located between two independently folded domains. The enzyme engages substrates when the active site nucleophile, Thr85, is phosphorylated | Bacillus cereus |