BRENDA - Enzyme Database show

Crystallization of human nicotinamide phosphoribosyltransferase

Takahashi, R.; Nakamura, S.; Yoshida, T.; Kobayashi, Y.; Ohkubo, T.; Acta Crystallogr. Sect. F 63, 375-377 (2007)

Data extracted from this reference:

Application
EC Number
Application
Commentary
Organism
2.4.2.12
drug development
inhibitor-soaking experiments
Homo sapiens
Cloned(Commentary)
EC Number
Commentary
Organism
2.4.2.12
cDNA into pGEX6p1 for expression in Escherichia coli BL21 (DE) as glutathione S-transferase (GST) tagged fusion protein
Homo sapiens
Crystallization (Commentary)
EC Number
Crystallization
Organism
2.4.2.12
apo enzyme, diffraction of third crystals at 2 A resolution at -174°C, crystals belong to space group P2(1) with unit-cell parameters a = 60.56 A, b = 106.40 A, c =82.78 A, beta = 96.50°, technique: 1 microlitre of 10 mg/ml protein solution to 1 microlitre reservoir solution (38% pentaerytritol propoxylate (5/4 PO/OH) and 200 mM KCl pH 9) one week at 4°C, generation of first crystals by hanging-drop vapour diffusion (Index HT screening kit), generation of second crystals by microseeding: reservoir solution contained crushed first crystals, generation of third crystals by Repeated Seeding Technique: reservoir solution contained crushed second crystals
Homo sapiens
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl-1-diphosphate
Homo sapiens
-
nicotinamide mononucleotide + diphosphate
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.4.2.12
Homo sapiens
P43490
-
-
Purification (Commentary)
EC Number
Commentary
Organism
2.4.2.12
lysis of bacteria by ultrasonification in presence of 1 mM phenylmethylsulfonylfluorid (PMSF) and 1 mM dithiothreitol (DTT), ultracentrifugation, supernatant applied to glutathione Sepharose 4B beads and washed, cleavage of glutathione S-transferase (GST) tag by PreScission Protease, purification by gel-filtration chromatography in Tris-HCl pH 8.5, 150 mM NaCl, 1 mM DTT, concentration to 10 mg/ml by ultrafiltration on Microcon YM-10, stored at 4°C
Homo sapiens
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl-1-diphosphate
-
690246
Homo sapiens
nicotinamide mononucleotide + diphosphate
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
2.4.2.12
dimer
according to sedimentation-equilibrium measurements following ultracentrifugation of the protein solution
Homo sapiens
2.4.2.12
homodimer
two molecules in the asymmetric unit of the crystal according to Matthews coefficient Vm = 2.4 A/Da, corresponding to a solvent content of 48.5%
Homo sapiens
Application (protein specific)
EC Number
Application
Commentary
Organism
2.4.2.12
drug development
inhibitor-soaking experiments
Homo sapiens
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.4.2.12
cDNA into pGEX6p1 for expression in Escherichia coli BL21 (DE) as glutathione S-transferase (GST) tagged fusion protein
Homo sapiens
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.4.2.12
apo enzyme, diffraction of third crystals at 2 A resolution at -174°C, crystals belong to space group P2(1) with unit-cell parameters a = 60.56 A, b = 106.40 A, c =82.78 A, beta = 96.50°, technique: 1 microlitre of 10 mg/ml protein solution to 1 microlitre reservoir solution (38% pentaerytritol propoxylate (5/4 PO/OH) and 200 mM KCl pH 9) one week at 4°C, generation of first crystals by hanging-drop vapour diffusion (Index HT screening kit), generation of second crystals by microseeding: reservoir solution contained crushed first crystals, generation of third crystals by Repeated Seeding Technique: reservoir solution contained crushed second crystals
Homo sapiens
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl-1-diphosphate
Homo sapiens
-
nicotinamide mononucleotide + diphosphate
-
-
r
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.4.2.12
lysis of bacteria by ultrasonification in presence of 1 mM phenylmethylsulfonylfluorid (PMSF) and 1 mM dithiothreitol (DTT), ultracentrifugation, supernatant applied to glutathione Sepharose 4B beads and washed, cleavage of glutathione S-transferase (GST) tag by PreScission Protease, purification by gel-filtration chromatography in Tris-HCl pH 8.5, 150 mM NaCl, 1 mM DTT, concentration to 10 mg/ml by ultrafiltration on Microcon YM-10, stored at 4°C
Homo sapiens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.4.2.12
nicotinamide + alpha-D-5-phosphoribosyl-1-diphosphate
-
690246
Homo sapiens
nicotinamide mononucleotide + diphosphate
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
2.4.2.12
dimer
according to sedimentation-equilibrium measurements following ultracentrifugation of the protein solution
Homo sapiens
2.4.2.12
homodimer
two molecules in the asymmetric unit of the crystal according to Matthews coefficient Vm = 2.4 A/Da, corresponding to a solvent content of 48.5%
Homo sapiens