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Literature summary extracted from

  • Iverson, T.M.; Panosian, T.D.; Birmingham, W.R.; Nannemann, D.P.; Bachmann, B.O.
    Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase (2012), Biochemistry, 51, 1964-1975.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
5.4.2.7 glucose 1,6-bisphosphate required for activation. The conformational change in Lys240 alters the affinity of the enzyme for the activator Bacillus cereus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.4.2.7 expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Bacillus cereus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
5.4.2.7 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

EC Number Protein Variants Comment Organism
5.4.2.7 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
5.4.2.7 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
5.4.2.7 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
5.4.2.7 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

EC Number General Stability Organism
5.4.2.7 the phosphoenzyme is stable throughout purification and crystallization Bacillus cereus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
5.4.2.7 Mn2+ a di-Mn2+ enzyme Bacillus cereus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.4.2.7 alpha-D-ribose 1-phosphate Bacillus cereus
-
 alpha-D-ribose 5-phosphate
-
 r
5.4.2.7 alpha-D-ribose 1-phosphate Bacillus cereus DSM 31
-
 alpha-D-ribose 5-phosphate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
5.4.2.7 Bacillus cereus Q818Z9
-
-
5.4.2.7 Bacillus cereus DSM 31 Q818Z9
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
5.4.2.7 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 (Commentary)

EC Number Purification (Comment) Organism
5.4.2.7 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.4.2.7 alpha-D-ribose 1-phosphate
-
 Bacillus cereus alpha-D-ribose 5-phosphate
-
 r
5.4.2.7 alpha-D-ribose 1-phosphate
-
 Bacillus cereus DSM 31 alpha-D-ribose 5-phosphate
-
 r

Subunits

EC Number Subunits Comment Organism
5.4.2.7 More the active site is located between two independently folded domains Bacillus cereus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.4.2.7 20 22 assay at Bacillus cereus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.4.2.7 8
-
 assay at Bacillus cereus

General Information

EC Number General Information Comment Organism
5.4.2.7 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