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Literature summary for 2.5.1.17 extracted from

  • Tanaka, Y.; Sasaki, T.; Kumagai, I.; Yasutake, Y.; Yao, M.; Tanaka, I.; Tsumoto, K.
    Molecular properties of two proteins homologous to PduO-type ATP:cob(I)alamin adenosyltransferase from Sulfolobus tokodaii. (2007), Proteins, 68, 446-457.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli as a 6His-tagged fusion protein Sulfurisphaera tokodaii
expression in Escherichia coli as a His-tagged protein Sulfurisphaera tokodaii
from genomic DNA in pET20b for expression with N-terminal hexa-His tag in Escherichia coli strain BL21 (DE3) Sulfurisphaera tokodaii
from genomic DNA in pET20b for expression with N-terminal hexa-His tag in Escherichia coli strain BL21 (DE3) or B834 (DE3) for the selenomethionine derivative Sulfurisphaera tokodaii

Crystallization (Commentary)

Crystallization (Comment) Organism
3fold symmetric trimer of five counterclockwise helix bundles, one molecule in asymmetric unit, polypropylene glycol 400 molecule captured in putative active site (positively charged, important residues: Asp32, Arg118), crystals of selenomethionine derivative: space group P2(1)3, unit cell parameters: a, b, c: 84.67 A, hanging-drop vapour-diffusion method: protein solution (15 mg/ml) + reservoir solution (pH 8.1, 2.5 M ammonium sulphate, 2% polypropylene glycol 400) Sulfurisphaera tokodaii
hanging-drop vapor diffusion method Sulfurisphaera tokodaii
sparse matrix method at 20°C Sulfurisphaera tokodaii
trimer with noncrystallographic 3fold symmetry in the asymmetric unit, consistent of five helix bundles, identical topology to ST1454 but less ion pairs around the putative active site, crystals: space group P4(1)2(1)2, unit cell parameters: a, b: 117.58 A, c: 79.05 A, hanging-drop vapour-diffusion method: protein solution (21 mg/ml) + reservoir solution (pH6.8, 0.5 M ammonium sulfate, 2% polypropylene glycol 400), molecular replacement Sulfurisphaera tokodaii

General Stability

General Stability Organism
secondary structure is retained even in 7 M guanidine hydrochroride Sulfurisphaera tokodaii
secondary structure stable even at 7 M guanidine hydrochloride as revealed by circular dichroism spectrophotometry, stability may be due to high numbers of ion pairs and ion pair networks around the active site (especially Glu125) and 3fold axis Sulfurisphaera tokodaii
secondary structure starts melting in presence of 3.5 M guanidine hydrochloride and is completely denatured at 4.5 M as revealed by circular dichroism spectrophotometry, possibly due to lower numbers of ion pairs and ion pair networks around the putative active site and 3fold axis compared to homolog ST1454 Sulfurisphaera tokodaii

Localization

Localization Comment Organism GeneOntology No. Textmining
soluble
-
Sulfurisphaera tokodaii
-
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
additional information
-
no extra band of recombinant hexa-His tagged protein in Western blotting Sulfurisphaera tokodaii
17890
-
MALDI-TOF analyses of recombinant hexa-His tagged protein, N-terminal deletion due to cleavage between Thr12 and Lys13, proportion of this species increases over time Sulfurisphaera tokodaii
19200
-
theoretical, deduced from primary sequence Sulfurisphaera tokodaii
19230
-
matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy Sulfurisphaera tokodaii
19230
-
MALDI-TOF analyses of recombinant hexa-His tagged protein, N-terminal deletion due to cleavage between Thr11 and Asn12 Sulfurisphaera tokodaii
20000
-
3 * 20000, revealed by size-exclusion chromatography and crystal structure analysis Sulfurisphaera tokodaii
20400
-
theoretical, deduced from primary sequence Sulfurisphaera tokodaii
60000
-
extra band of recombinant hexa-His tagged protein in Western blotting Sulfurisphaera tokodaii

Organic Solvent Stability

Organic Solvent Comment Organism
guanidine-HCl 7 M, activity is retained Sulfurisphaera tokodaii

Organism

Organism UniProt Comment Textmining
Sulfurisphaera tokodaii
-
thermophilic archeon
-
Sulfurisphaera tokodaii Q970Z7
-
-
Sulfurisphaera tokodaii 7 Q970Z7
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Sulfurisphaera tokodaii
from bacterial lysate by heat treatment (30 min, 70°C) and chromatography: HiTrap Phenyl FF column (2-0 M NaCl gradient, pH 8) and HiLoad 26/60 Superdex 75-pg column (pH 8), for the selenomethionine derivative: HiTrap QXL column (0-2 M NaCl gradient, pH 9) and HiLoad 26/60 Superdex 75-pg column (pH 8) Sulfurisphaera tokodaii
from bacterial lysate by heat treatment (30 min, 70°C) and chromatography: HiTrap QXL column (0-2 M NaCl gradient, pH9) and HiLoad 26/60 Superdex 75-pg column (pH8) Sulfurisphaera tokodaii

Reaction

Reaction Comment Organism Reaction ID
2 ATP + 2 cob(II)yrinic acid a,c-diamide + a reduced flavoprotein = 2 triphosphate + 2 adenosylcob(III)yrinic acid a,c-diamide + an oxidized flavoprotein not yet confimed Sulfurisphaera tokodaii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + cob(I)alamin
-
Sulfurisphaera tokodaii tripolyphosphate + coenzyme B12
-
?
ATP + cob(I)alamin
-
Sulfurisphaera tokodaii 7 tripolyphosphate + coenzyme B12
-
?
ATP + cob(I)alamin + H2O anaerobic, 20 min, 80°C, pH 8, in presence of 1 mM titanium (III) citrate Sulfurisphaera tokodaii ? ATP-dependent cob(I)alamin consumption to a yet unknown compound ?
ATP + cob(I)alamin + H2O anaerobic, 20 min, 80°C, pH 8, in presence of 1 mM titanium (III) citrate Sulfurisphaera tokodaii phosphate + diphosphate + adenosylcobalamin measured by decrease in light absorbance by cob(I)alamin at 388 nm and increase of light absorbance by presumably adenosylcobalamin at 525 nm ?
additional information indications for cob(I)alamin-binding due to decrease in light absorbance by cob(I)alamin at 388 nm and conversion to a compound with absorbance maximum at 485 nm, different from adenosylcobalamin Sulfurisphaera tokodaii ?
-
?
additional information no ATP: cob(I)alamin adenosyltransferase activity detectable (anaerobic, 20 min, 80°C, pH 8, in presence of 1 mM titanium (III) citrate), no increase in light absorbance by presumably adenosylcobalamin at 525 nm Sulfurisphaera tokodaii ?
-
?

Subunits

Subunits Comment Organism
? x * 19230, recombinant enzyme with a 6His-tag, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy. The 6His-tagged recombinant enzyme can exist as an oligomer in SDS-PAGE Sulfurisphaera tokodaii
trimer 3 * 20000, revealed by size-exclusion chromatography and crystal structure analysis Sulfurisphaera tokodaii

Synonyms

Synonyms Comment Organism
ATP:cob(I)alamin adenosyltransferase
-
Sulfurisphaera tokodaii
homologeous to PduO-type ATP: cob(I)alamin adenosyltransferase
-
Sulfurisphaera tokodaii
ST1454
-
Sulfurisphaera tokodaii
ST1454 locus name Sulfurisphaera tokodaii
ST2180
-
Sulfurisphaera tokodaii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
80
-
assay at Sulfurisphaera tokodaii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Sulfurisphaera tokodaii