Information on EC 2.5.1.17 - cob(I)yrinic acid a,c-diamide adenosyltransferase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea

EC NUMBER
COMMENTARY hide
2.5.1.17
-
RECOMMENDED NAME
GeneOntology No.
cob(I)yrinic acid a,c-diamide adenosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
adenosyl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I
-
-
adenosylcobalamin biosynthesis from cobyrinate a,c-diamide II
-
-
adenosylcobalamin salvage from cobalamin
-
-
adenosylcobalamin salvage from cobinamide I
-
-
adenosylcobalamin salvage from cobinamide II
-
-
Metabolic pathways
-
-
Porphyrin and chlorophyll metabolism
-
-
vitamin B12 metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:cob(I)yrinic acid-a,c-diamide Cobeta-adenosyltransferase
The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) to Co(II) by a one-electron transfer. This can be carried out by EC 1.16.1.3, aquacobalamin reductase or non-enzymically in the presence of dihydroflavin nucleotides [2]. (ii) Co(II) is reduced to Co(I) in a second single-electron transfer by EC 1.16.1.4, cob(II)alamin reductase and (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP to leave the cobalt atom in a Co(III) state (EC 2.5.1.17). The enzyme responsible for the adenosylation reaction is the product of the gene cobO in the aerobic bacterium Pseudomonas denitrificans and of the gene cobA in the anaerobic bacterium Salmonella typhimurium. In P. denitrificans, the enzyme shows specificity for cobyrinic acid a,c-diamide and the corrinoids that occur later in the biosynthetic pathway whereas CobA seems to have broader specificity [3]. While CobA has a preference for ATP and Mn2+, it is able to transfer a variety of nucleosides to the cobalt, including CTP, UTP and GTP, in decreasing order of preference [4] and to use Mg2+ instead of Mn2+.
CAS REGISTRY NUMBER
COMMENTARY hide
37277-84-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
strain CRL1098, gene pduO
-
-
Manually annotated by BRENDA team
strain Go1, orf MM3138, housekeeping gene cobAMm
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-
Manually annotated by BRENDA team
recombinant strain SC510 Rifr containing plasmid pXL227, this DNA seuqence encodes for several enzymes
SwissProt
Manually annotated by BRENDA team
HH103, strain SVQ336; HH103, strain SVQ524
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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a HH103 cobO mutant (strain SVQ524), is constructed by the insertion of omega interposon in the BglII site of cobO gene. Mutant is auxotroph for methionine and cobalamin as it is shown that the presence of either compound restores the growth on minimal medium. Mutant SVQ524 fails to nodulate on Vigna radiate but is able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan. The roots of mutant plants do not secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine auxotrophs in the rhizosphere. The phenotype of SVQ524 is rescued by the addition of methionine or cobalamin to the plant growth media or by the presence of a copy of the cobO gene; a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 is obtained by Tn5-lacZ mutagenesis. Sequence analysis show that the transposon is inserted into a gene homologous to cobO. cobO mutant SVQ336 is auxotroph for methionine and cobalamin as it is shown that the presence of either compound restores the growth of SVQ336 on minimal medium
physiological function
-
ATP:co(I)rrinoid adenosyltransferases are enzymes that catalyze the formation of adenosylcobalamin, i.e. coenzyme B12, from cobalamin and ATP. CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond. In Salmonella enterica, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2'-deoxy-ATP + cob(I)alamin + H2O
?
show the reaction diagram
-
-
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(I)alamine
show the reaction diagram
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
ATP + cob(I)alamin
tripolyphosphate + coenzyme B12
show the reaction diagram
ATP + cob(I)alamin + H2O
?
show the reaction diagram
anaerobic, 20 min, 80C, pH 8, in presence of 1 mM titanium (III) citrate
ATP-dependent cob(I)alamin consumption to a yet unknown compound
-
?
ATP + cob(I)alamin + H2O
adenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
monitoring adenosylcobalamin formation at 388 nm in continous spectrophotometric assay
-
?
ATP + cob(I)alamin + H2O
phosphate + diphosphate + adenosylcobalamin
show the reaction diagram
anaerobic, 20 min, 80C, pH 8, in presence of 1 mM titanium (III) citrate
measured by decrease in light absorbance by cob(I)alamin at 388 nm and increase of light absorbance by presumably adenosylcobalamin at 525 nm
-
?
ATP + cobalamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
enzyme is absolutely specific for ATP or dATP as adenosyl donors, ATP is the preferred adenosyl donor
-
-
?
ATP + cobalamin
triphosphate + adenosylcobalamin
show the reaction diagram
enzyme is absolutely specific for ATP or dATP as adenosyl donors
-
-
ir
ATP + cobinamide
triphosphate + adenosylcobinamide
show the reaction diagram
-
-
-
-
?
ATP + hydroxycobalamin
adenosylcobalamin + phosphate + diphosphate
show the reaction diagram
-
37C, pH 8, 0.5 mM ATP, 0.05 mM hydroxycobalamin, in presence of 1 mM titanium(III)citrate
measured by decrease in absorbance at 388 nm
-
?
ATP + hydroxycobalamin
tripolyphosphate + adenosylcobalamin + H2O
show the reaction diagram
-
coenzyme B12 synthesis from vitamin B12, dimethylbenzimidazole arm of vitamin B12 plays no role in substrate positioning, corrinoid adenosylation assay: anaerobic, pH 6, 25C, 1 or 2 mM FMN, 10 or 20 mM NADH, NAD(P)H: flavin oxidoreductase, 2 h incubation for complete reduction of hydroxycobalamin to cob(II)alamin before initiation of adenosyltransfer
measuring difference in absorbance by adenosylcobalamin at 525 nm
-
?
cob(I)alamin + ADP
adenosylcobalamin + diphosphate
show the reaction diagram
cob(I)alamin + ATP
adenosylcobalamin + ?
show the reaction diagram
-
in the co+ assay the cobalt ion of cobalamin is chemically reduced in solution to cob(I)alamin by Ti(III)citrate, allowing the cob(I)alamin adenosylation reaction to be measured directly. The Co+ assay is performed under anoxic conditions
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
cob(I)alamin + ATP + H2O + H+
adenosylcobalamin + diphosphate + phosphate
show the reaction diagram
cob(I)alamin + CTP + H2O + H+
cytosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + dATP + H2O + H+
deoxyadenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + GTP + H2O + H+
guanosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + ITP
hypoxanthosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + UTP
uranylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)inamide + ATP
5'-deoxy-5'-adenosyl-cob(I)inamide + polyphosphate
show the reaction diagram
-
-
-
cob(I)inamide + ATP
adenosylcobinamide + triphosphate
show the reaction diagram
cob(I)yric acid + ATP
5'-deoxy-5'-adenosyl-cob(I)yric acid + polyphosphate
show the reaction diagram
-
-
-
cob(I)yrinic acid a,c-diamide + ATP
5'-deoxy-5'-adenosyl-cob(I)yrinic acid a,c-diamide + polyphosphate
show the reaction diagram
-
-
-
cob(II)alamin
cob(I)alamin
show the reaction diagram
-
G97, T161, and H183 possible role in stabilizing four-coordinate, cob(II)alamin C-terminal His-tagged enzyme binds cob(II)alamin base-off while N-terminal His-tagged enzyme binds it base-on (impaired base-off transition), only mutants S68F, K78Q, K78R, R186W, and R190C also bind cob(II)alamin base-off
-
-
?
cob(II)alamin + ATP
adenosylcobalamin + ?
show the reaction diagram
-
in the Co2+ assay the NADPH-dependent flavodoxin protein reductase/flavodoxin system is used to reduce Co2+ to Co+. In this Co2+ assay, the PduO enzyme must bind cob(II)alamin and facilitate the generation of cob(I)alamin in its active site
-
-
?
CTP + cob(I)alamin
triphosphate + cytosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, 9% activity compared to ATP with enzyme variant 239K, 6% activity compared to ATP with enzyme variant 239M
-
-
?
CTP + cob(I)alamin + H2O
cytidylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
cyanocob(I)alamin + ATP
tripolyphosphate + alpha-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide
show the reaction diagram
dATP + cob(I)alamin
tripolyphosphate + deoxyadenosylcobalamin
show the reaction diagram
dATP + cobalamin
triphosphate + deoxyadenosylcob(III)alamin
show the reaction diagram
-
enzyme is absolutely specific for ATP or dATP as adenosyl donors, dATP results in 21% of the activity with ATP
-
-
?
dATP + cobalamin
triphosphate + deoxyadenosylcobalamin
show the reaction diagram
enzyme is absolutely specific for ATP or dATP as adenosyl donors
-
-
ir
GTP + cob(I)alamin
triphosphate + guanosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, 16% activity compared to ATP with enzyme variant 239K, 14% activity compared to ATP with enzyme variant 239M
-
-
?
GTP + cob(I)alamin + H2O
guanosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
ITP + cob(I)alamin + H2O
inosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
UTP + cob(I)alamin
triphosphate + uridylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, 8% activity compared to ATP with enzyme variant 239K, 6% activity compared to ATP with enzyme variant 239M
-
-
?
UTP + cob(I)alamin + H2O
uridylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + cob(I)alamin
triphosphate + adenosylcob(I)alamine
show the reaction diagram
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
ATP + cobinamide
triphosphate + adenosylcobinamide
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ADP
adenosylcobalamin + diphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
cob(I)alamin + ATP + H2O + H+
adenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + CTP + H2O + H+
cytosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + dATP + H2O + H+
deoxyadenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + GTP + H2O + H+
guanosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + ITP
hypoxanthosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + UTP
uranylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)inamide + ATP
adenosylcobinamide + triphosphate
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CTP
-
about 40% activity compared to ATP
dATP
-
about 25% of the activity with ATP
flavodoxin A
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in vitro reduced flavodoxin provides an electron to generate the co(I)rrinoid substrate in the CobA active site, modeling of enzyme ligand interaction, residues R9 and R165 are important for CobA-FldA docking but not to catalysis, overview
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GTP
-
about 5% activity compared to ATP
ITP
-
about 8% activity compared to ATP
UTP
-
about 10% activity compared to ATP
additional information
-
no activity with AMP as cofactor
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
activates 2.5fold
Fe2+
-
required and contained by EutT
Ni2+
-
1.5fold activation
titanium citrate
-
-
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxyadenosine
-
weak inhibition
5'-mercapto-5'-deoxyadenosine-5'-triphosphate
-
complete inhibition at a 3 mM concentration
adenine
-
weak inhibition
adenosine
-
weak inhibition
ADP
-
about 90% inhibition
bathophenanthroline
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inhibition of EutT
cob(I)alamin
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substrate inhibition at more than 0.008 mM in presence of subsaturating concentrations of ATP (0.003 mM)
cobinamide
Cu2+
-
complete inhibition
diphosphate
hydrogenobyrinic acid a,c-diamide
-
cobalt-free analogue of cobyrinic acid a,c-diamide
Triphosphate
tripolyphosphate
TWYYGEAQCDWDD68
-
competitive 30% inhibition of flavodoxin-dependent adenosylcobalamin synthesis by a FldA peptide TWYYGEAQCDWDD68, the inhibition depends on residues Glu61 and Asp68, overview, no inhibition by peptides TWYYGAAQCDWDA68 and WPTAGYHFEASKG132
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
-
induces formation of activated Co2+ species
coenzyme B12
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0016
2'-deoxy-ATP
-
-
0.0003 - 1.3
ATP
0.27
Co2+
pH 8.0, 70C, wild-type enzyme
0.00007 - 0.06
cob(I)alamin
0.000096
cob(I)inamide
-
-
0.0078 - 0.134
cob(II)alamin
0.003 - 0.004
Cobalamin
0.01
cyanocob(I)alamin
0.14
dATP
0.003
hydroxocobalamin
0.11
Mg2+
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0051
2'-deoxy-ATP
Lactobacillus reuteri
-
-
0.00038 - 297.6
ATP
0.00041 - 297.6
cob(I)alamin
0.02
cob(I)inamide
Lactobacillus reuteri
-
-
0.00067 - 0.038
cob(II)alamin
0.11 - 0.18
Cobalamin
0.0086
CTP
Lactobacillus reuteri
-
-
0.11
dATP
0.021
GTP
Lactobacillus reuteri
-
-
0.18
hydroxocobalamin
0.019
ITP
Lactobacillus reuteri
-
-
0.00195
UTP
Lactobacillus reuteri
-
-
additional information
2'-deoxy-ATP
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.057 - 200
ATP
0.4 - 395
cob(I)alamin
210
cob(I)inamide
Lactobacillus reuteri
-
-
22100
0.013 - 4.8
cob(II)alamin
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0013
TWYYGEAQCDWDD68
-
37C, recombinant wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000125
-
activity measured using washed ribosomes; pH 8.0, 37C
0.0005
-
in presence of 1 mM ATP, 20 mM NADH, 2 mM FMN, 0.5 mM hydroxycobalamin and NAD(P)H: flavin oxidoreductase, 1.5 mM MgCl2, 100 mM KCl, pH 6
0.001
-
purified enzyme, pH 8.0
0.0061
0.02
-
purified recombinant enzyme
0.023
-
reductant: PduS protein, specific activity of LrPduO using different electron sources for the adenosylation of cob(II)alamin
0.026
-
reductant: PduS protein, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.027
-
purified recombinant GST-tagged mutant R191W
0.034
recombinant mutant R124F
0.045
-
reductant: NADPH-dependent ferredoxin protein reductase, specific activity of LrPduO using different electron sources for the adenosylation of cob(II)alamin
0.047
-
bovine ATR containing mitochondrial targeting sequence, expressed in E. coli, soluble fraction; pH 8.0, 37C
0.053
-
activity at final purification step; pH 8.0, 37C
0.061
-
human ATR containing mitochondrial targeting sequence, expressed in E. coli and forming inclusion bodies; pH 8.0, 37C
0.072
-
reductant: NADPH-dependent ferredoxin protein reductase, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.091
-
purified recombinant full length wild-type enzyme, in absence of Mg2+
0.098
-
human ATR with no mitochondrial targeting sequence, expressed in E. coli and forming inclusion bodies; pH 8.0, 37C
0.13
-
in the presence of cobyrinic acid a,c-diamide as substrate; pH 8.0, 30C
0.143
-
purified recombinant wild-type enzyme
0.18
-
in the presence of cobinamide as substrate; pH 8.0, 30C
0.19
-
recombinant enzyme variant 239M
0.22
-
recombinant enzyme variant 239K
0.262
-
purified recombinant full length wild-type enzyme, in presence of 2.5 mM MgCl2
0.312
activity of inclusion bodies formed with enzyme expressed in E.coli; pH 8.0, 37C
0.385
recombinant mutant R124A
0.49
-
reductant: Ti(III)citrate, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.619
recombinant wild-type enzyme
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.5
-
30% of maximal activity at pH 7.5
7.5 - 8.5
-
75% of maximal activity at pH 7.5, 94% at pH 8.5
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
skin fibroblasts show low abundance
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
subcellular distribution
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus cereus (strain ATCC 14579 / DSM 31 / JCM 2152 / NBRC 15305 / NCIMB 9373 / NRRL B-3711)
Bacillus cereus (strain ATCC 14579 / DSM 31 / JCM 2152 / NBRC 15305 / NCIMB 9373 / NRRL B-3711)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
19200
theoretical, deduced from primary sequence
19230
MALDI-TOF analyses of recombinant hexa-His tagged protein, N-terminal deletion due to cleavage between Thr11 and Asn12; matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy
20400
theoretical, deduced from primary sequence
36600
calculated from amino acid sequence
37000
SDS-PAGE of recombinant enzyme expressed in E. coli
44000
-
gel filtration
52000
-
SDS-PAGE, recombinant enzyme without mitochondrial targeting sequence
54000
-
calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence
55000
-
calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence
60000
extra band of recombinant hexa-His tagged protein in Western blotting
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
trimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apoenzyme and in complex with Mg2+/ATP, C-centred orthorhombic space group C222(1), unit cell parameters: a: 64.93 A, b: 137.08 A, c: 158.55 A, alpha, beta, gamma: 90, one trimer in the asymmetric unit, sitting-drop combined with hanging-drop vapour-diffusion method: 13 mg/ml protein solution, precipitants: 1.55-1.6 M ammonium sulfate, 9-10% (v/v) dioxane (pH 6.5), for complex: 4 mM ATP and 4 mM Mg2+
-
purified recombinant PduO in complex with ATP, hanging drop vapor diffusion method, mixing o 0.001 ml of 13 mg/ml protein solution, with or without 4 mM ATP and 4 mM MgCl2, with 0.001 ml of optimized reservoir solution containing 100 mM MES, pH 6.5, 1.52 M ammonium sulfate, 9% v/v dioxane, followed by equilibration over 0.5 ml of the mother liquor, the cryoprotection solution contains 50 mM MES, pH 6.5, 0.76 M ammonium sulfate, 4.5% v/v dioxane, and 1.7 M sodium malonate, pH 7.0, X-ray diffraction structure determination and analysis
-
native (PDB: 2ZHY, with ordered N-terminal loop and formed active site) and in complex with ATP (PDB: 2ZHZ, substrate-binding cleft is widened and the N-terminal loop swung out and conformational shift of Arg129 side chain upon ATP-binding, similar structure to human and Lactobacillus reuteri PduO except for interaction of NH2 nitrogen atom of Arg11 with gamma-phosphate of ATP), five alpha helix bundle, monomeric subunits almost identical conformations in both structures, crystals: orthothrombic space group C222(1), three monomers in the asymmetric unit, unit cell parameters: a: 52.55/53.91, b: 148.98/148.17, c: 157.35/158.10, microcrystals (from sitting-drop vapour-diffusion at 22C) scaled up by hanging-drop vapour diffusion, reservoir solution (pH5.7, 22% (w/v) isopropanol, 12% (v/v) PEG4000), PduO-MgATP complex: ATP soaked into native PduO crystals, molecular replacement using PDB: 2G2D as model
mutants D35N (without tag) in complex with ATP and cob(II)alamin and R132K (without tag) in complex with ATP, thin plate crystals, space group P6(3), two monomers in the asymmetric unit, unit cell parameters: a, b: 65A, c: 169A, beta: 90; vapour-diffusion under anoxic conditions, protein solution (15 mg/ml, containing ATP, hydroxycobalamin and a reducing system of NADH, FMN, and flavodoxin reductase), reservoir solution (incl. 14-16% PEG8000, pH6)
-
purified recombinant His-tagged enzyme in complex with its substrates, hanging drop vapour diffusion method, 20C, 0.004 ml of 20 mg/ml protein in 10 mM Tris-HCl, pH 8.0, is mixed with 0.004 ml od precipitant solution containing 0.1 M HEPES, pH 8.5, 1.1 M ammonium sulfate, 2 mM ATP, 55 mM MgCl2, 165 mM NaCl, and 15 mM cob(I)alamin, 7 days, X-ray diffraction structure determination and analysis at 1.68 A resolution
-
trimer of three independent five-helix bundles, active sites at the interface between adjacent monomers, no significant structural changes accompany catalysis, precatalytic complex with ATP: cob(II)alamin (PDB: 3CI1, four-coordinate, base-off cob(II)alamin intermediate, enzyme with fully ordered six C-terminal residues and potassium ion in active site), complex with tripolyphosphate: adenosylcobalamin (PDB: 3CI3, partially occupied with five-coordinate adenosylcobalamin), precatalytic complex with ATP: cob(II)inamide (PDB: 3CI4, cob(II)inamide-binding structurally indistinguishable from cob(II)alamin-binding), binding of cobalamin and cobinamide (lacking dimethylbenzimidazole moiety) in identical positions and orientation, space group R3, one molecule in asymmetric unit, unit cell parameters: a: 67.8-68, b: 67.8-68, c: 110.9-111.3, beta: 90, molecular replacement using PDB: 2NT8 as model; vapour-diffusion with tag-cleaved protein solution (18-22 mg/ml, in presence of hydroxycobalamin and/or adenosylcobalamin or dicyanocobinamide, ATP etc.) and reservoir solution (10-13% (w/v) PEG 8000, pH 6), cubic crystals, crystallisation under anoxic conditions in presence of flavin-dependent reducing system
-
purified recombinant CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin, hanging drop vapour diffusion method, 0.002 ml of 10 mg/ml CobA protein in 20 mM Tris-HCl, pH 8.0, 20 mM NADH, 3 mM ATP, 4.5 mM MgCl2, and 2 mM HOCbl, is mixed with 0.002 ml of well solution containing 100 mM MES, pH 6.0, 320 mM NaCl, and 19.6% w/v PEG4000, X-ray diffraction structure determination and analysis at 1.95 A resolution
-
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); hanging-drop vapor diffusion method; sparse matrix method at 20C; 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
purified recombinant wild-type and selenomethionine-labeled enzymes, crystal growth from 0.4 M ammonium phosphate, 4% methyl-pentanediol, 5% glycerol, at 20C, X-ray diffraction structure determination and analysis at 1.5-1.9 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
stable for several weeks with dithiothreitol
55
-
sharp decrease in activity with preincubation at 55C or higher
60
-
no appreciable loss of activity after 10 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol stabilizes during purification
2-mercaptoethanol stabilizes the enzyme during purfication
dithiothreitol stabilizes the enzyme
-
DTT stabilizes during purification and storage, removal leads to complete loss of activity
-
repeated freeze-thawing inactivates
secondary structure is retained even in 7 M guanidine hydrochroride
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
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
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
guanidine-HCl
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is oxygen-labile
-
658993
EutT is oxygen-labile, exposure to air leads to complete loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, lyophilized, at least 1 week
-
-20C, more than 50% loss of activity within 1 week, with 50% glycerol only 20% loss of activity
-
2-mercaptoethanol stabilizes during storage
4C, about 1 month
-
DTT stabilizes during storage
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 recombinant polymorphic variants 239K and 239M of the enzyme from Escherichia coli in a multistep procedure to homogeneity, 4.5- and 5.4fold, respectively
-
; from bacterial lysate by heat treatment (30 min, 70C) 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); from bacterial lysate by heat treatment (30 min, 70C) and chromatography: HiTrap QXL column (0-2 M NaCl gradient, pH9) and HiLoad 26/60 Superdex 75-pg column (pH8)
after expression at 16C, lysis with French pressure cell, pH 7.5, and centrifugation
-
ammonium sulfate precipitation followed by two chromatographic steps
-
centrifugation and SDS-PAGE
-
centrifugation, Mono Q chromatography and gel filtration
-
from crude lysate by HiTrap Ni2+-chelating affinity chromatography (gradient elution), gel filtration on HiLoad 16/60 Superdex 200 (pH7.9, 4 mM dihiothreitol), concentration to 15 mg/ml by ultrafiltration, yield: 45 mg pure protein per litre culture
native enzyme by anion exchange chromatography, dialysis, and hydrophobic interaction chromatography, recombinant enzyme from Escherichia coli strain BL21(DE3)
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nickel-chelating HiTrap chromatography (elution with 1 M imidazole), HiLoad 16/60 Superdex 200 gel filtration chromatography (2 mM dithiothreitol), concentration to 13 mg/ml by ultracentrifugation
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partial purification of the enzyme is achieved from ribosomal fraction by salt extraction and ammonium sulfate fractionation, followed by two chromatographic steps
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protamine sulfate, heat treatment and chromatography on Sephadex G-25 and DEAE-cellulose
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)
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recombinant His-tagged EutT from Escherichia coli to 70% purity
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
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recombinant His-tagged wild-type and mutant PduOs from Escherichia coli by nickel affinity chromatography to over 99% homogeneity
-
recombinant maltose-binding-protein or GST fusion wild-type and mutant enzymes from Escherichia coli strain DH5alpha by amylose and glutathione affinity chromatography, respectively
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recombinant PduO from Escherichia coli strain BL21 (DE3) cell culture medium
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recombinant solubilized enzyme from Escherichia coli 1.4fold to homogeneity, partial purification of recombinant truncated mutants, recombinant N-terminal domain to homogeneity
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recombinant tagless, wild-type CobA, and His7-tagged mutant enzymes by nickel affinity chromatography, the His-tag is cleaved off by rTEV protease
-
using a HisTrap FF column
using a HiTrap SP HP FPLC column and gel filtration
-
using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 enzymes encoded by genes eutT and cobA, DNA sequence determination, analysis of several geno- and phenotypic strains, enzyme is expressed e.g. as fusion protein or to complement a deficient strain, overview
; expression in Escherichia coli in strain BL21(DE3) of the wild-type enzyme and in strain B834(DE3) as selenomethionine-labeled enzyme
DNA sequence determination and analysis, expression of 2 polymorphic variants 239K and 239M of the enzyme in Escherichia coli as soluble proteins
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expressed in Escherichia coli
expressed in Escherichia coli as an N-terminal His-tagged fusion protein
expression in Escherichia coli as a 6His-tagged fusion protein; expression in Escherichia coli as a His-tagged protein; from genomic DNA in pET20b for expression with N-terminal hexa-His tag in Escherichia coli strain BL21 (DE3); 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
expression of the enzyme in C57/Bl6 mice using an adeno-associated virus vector, rAAV 2 and 8 vectors, with primer pairs specific to the cytomegalovirus enhancer/chicken beta-actin, CBAT, promoter, quantitative and semiquantitative expression analysis in murine liver, overview
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expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha as maltose-binding-protein or GST fusion protein
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expression of wild-type enzyme in Escherichia coli in inclusion bodies, expression of truncated mutants in Escherichia coli
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from genomic DNA in pET-28a for expression in Escherichia coli BL21 (DE3)
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from genomic DNA in pET28a for expression with a C-terminal hexa-His tag in Escherichia coli BL21 (DE3)
gene cobA, recombinant overexpression of tagless, wild-type CobA and expression of His7-tagged mutant CobAs in Escherichia coli strain BL21(lambdaDE3) carrying a null allele of btuR, the cobA homologue in this bacterium
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gene cobAMm, expression in a Salmonella enterica strain lacking the housekeeping CobA enzyme restores coenzyme B12 synthesis, overexpression in Escherichia coli strain BL21(DE3)
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gene eutT, overexpression of His-tagged enzyme in Escherichia coli as insoluble protein
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gene pduO, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene pduO, overexpression in Escherichia coli strain BL21 (DE3) and secretion to the medium
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gene pduO, recombinant overexpression of His-tagged wild-type and mutant PduOs in Escherichia coli
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overexpression of wild-type and mutant enzymes in Escherichia coli using strain BL21(DE3) for the His-tagged variant, and several derivatives of strain DH5alpha, overview
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QuickChangeXL site-directed mutagenesis performed on wild-type LrpduO in pTEV3 before expression of wild-type and variants in Escherichia coli BL21(DE3) with a rTEV protease-cleavable N-terminal hexa-His tag
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recombinantly expressed in Escherichia coli
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structural gene cobA
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structural gene cobO
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without mitochondrial targeting sequence from plasmid pNL166 harbouring the coding sequence, native or with C-terminal octa-His tag into pTA925 to introduce a linker region necessary for expression in Escherichia coli BL21DE3 RIL and ATR-deficient Salmonella strain BE620, hATR in pCF13 (N-terminal octa-His tag) and pCF15 (C-terminal octa-His tag) for transformation and random mutagenesis in Escherichia coli mutator strain XL1-Red followed by expression in BE620
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C119Y
-
wild-type kinetics, decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression, no rescue of ATR-deficient Salmonella strain BE620
C189Y
-
inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620
D218N
-
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
D64G
-
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
D90N
-
inactive in vitro (10fold excess of substrate compared to standard)
E84K
-
inactive in vitro (10fold excess of substrate compared to standard)
E91K
-
inactive in vitro (10fold excess of substrate compared to standard)
F212S
-
inactive in vitro (10fold excess of substrate compared to standard)
F83S
-
large change in KM for ATP and cob(I)alamin, F83 has direct contact with ATP
G63E
-
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
G87R
-
inactive in vitro (10fold excess of substrate compared to standard)
G97E
-
wild-type kinetics, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels
G97R
-
substantially reduced Vmax, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620
H183Y
-
wild-type kinetics, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels
K78Q
-
inactive in vitro (10fold excess of substrate compared to standard)
K78R
-
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
L220P
-
inactive in vitro (10fold excess of substrate compared to standard)
L223P
-
inactive in vitro (10fold excess of substrate compared to standard)
L92S
-
inactive in vitro (10fold excess of substrate compared to standard)
R190C
-
inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients
R190H
-
catalytically inactive patient mutation leading to the inherited disorder methylmalonic aciduria. Mutant is examined using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding. R190H and R186W significantly disrupt the affinity between MMAB and adenosylcobalmin. Arg 186 and Arg-190 may be critical for the transfer of the 5'-deoxyadenosyl group from ATP to cob(I)alamin, possibly by contributing to the precise positioning of the two substrates to permit catalysis to occur
R191W
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inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients
R191W/A135T
-
site-directed mutagenesis, the mutant enzyme shows 30% reduced activity compared to the wild-type enzyme
R194G
-
inactive in vitro (10fold excess of substrate compared to standard)
R215K
-
inactive in vitro (10fold excess of substrate compared to standard), lack of activity in vitro, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
R225K
-
lack of activity in vitro, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
R76G
-
large change in KM for ATP and cob(I)alamin, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
S126L
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large change in KM for ATP and cob(I)alamin, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
S217R
-
inactive in vitro (10fold excess of substrate compared to standard)
S68F
-
substantially reduced Vmax, residue S68 has role in ATP-binding
S94L
-
inactive in vitro (10fold excess of substrate compared to standard)
T161I
-
wild-type kinetics, decreased adenosylcobalamin production in vivo but rescues ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels
D35E/R128K
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reduced activity to lower extent than mutation R128K alone
D35N/R128K
-
similar kinetics as mutation D35N alone
D35R/R128D
-
reciprocal mutation to D35/R128, very high KM values did not allow for kinetic analyses at saturating substrate concentrations
DELTAS183
-
Km (mM) (Co+ assay): 0.0031 (ATP), 0.00007 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.023 (ATP), 0.02 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0099 (ATP), 0.0163 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.017 (ATP), 0.018 (cob(II)alamin)
F112W
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Km (mM) (Co+ assay): 0.002 (ATP), 0.00077 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.0018 (ATP), 0.002 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0073 (ATP), 0.028 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.0028 (ATP), 0.0032 (cob(II)alamin)
F112Y
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Km (mM) (Co+ assay): 0.0012 (ATP), 0.00055 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.0016 (ATP), 0.0017 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0094 (ATP), 0.034 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.0023 (ATP), 0.0027 (cob(II)alamin)
F163A
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Km (mM) (Co+ assay): 0.0159 (ATP), 0.00019 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.015 (ATP), 0.015 (cob(I)alamin), Km (mM) (Co2+ assay): 0.096 (ATP), 0.134 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.015 (ATP), 0.027 (cob(II)alamin)
S129A
-
mildly affected kcat and KM for both substrated
S159A
-
site-directed mutagenesis
V186A
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site-directed mutagenesis
A134L
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site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
C79A
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site-directed mutagenesis of EutT, the mutant shows highly reduced activity compared to the wild-type enzyme
C80A
-
site-directed mutagenesis of EutT, the mutant shows 99% reduced activity compared to the wild-type enzyme
C83A
-
site-directed mutagenesis of EutT, the mutant shows 99% reduced activity compared to the wild-type enzyme
F91A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91A,W93A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91D
-
site-directed mutagenesis, the mutant is inactive
F91H
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91W
-
site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme
F91W,W93F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91Y
-
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
R165A
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
R165E
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
R98A
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site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme
R98E
-
site-directed mutagenesis, the mutant shows about 30% reduced activity compared to the wild-type enzyme
R98E/R165E
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site-directed mutagenesis, the mutant shows 85% reduced activity compared to the wild-type enzyme
R9A
-
site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
R9E
-
site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
R9E/R165E
-
site-directed mutagenesis, the mutant shows 30% reduced activity compared to the wild-type enzyme
R9E/R98E
-
site-directed mutagenesis, the mutant shows 60% reduced activity compared to the wild-type enzyme
R9E/R98E/R165E
-
site-directed mutagenesis, the mutant shows 25% reduced activity compared to the wild-type enzyme
W93A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W93D
-
site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme
W93F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W93H
-
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
W93Y
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
insoluble recombinant enzyme is lysed under anoxic conditions using CHES/glycine buffer, pH 9.5, followed by resuspension of EutT from the pelleted material with 8 mM CHAPS, and centrifugation
-
solubilization of wild-type enzyme and truncated enzyme forms from inclusion bodies after expression in Escherichia coli
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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