Information on EC 1.1.98.6 - ribonucleoside-triphosphate reductase (formate)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.98.6
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RECOMMENDED NAME
GeneOntology No.
ribonucleoside-triphosphate reductase (formate)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ribonucleoside 5'-triphosphate + formate = 2'-deoxyribonucleoside 5'-triphosphate + CO2 + H2O
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Purine metabolism
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Pyrimidine metabolism
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Metabolic pathways
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guanosine deoxyribonucleotides de novo biosynthesis II
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superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli)
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adenosine deoxyribonucleotides de novo biosynthesis II
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pyrimidine deoxyribonucleotides de novo biosynthesis II
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SYSTEMATIC NAME
IUBMB Comments
ribonucleoside-5'-triphosphate:formate 2'-oxidoreductase
The enzyme, which is expressed in the bacterium Escherichia coli during anaerobic growth, contains an iron sulfur center. The active form of the enzyme contains an oxygen-sensitive glycyl (1-amino-2-oxoethan-1-yl) radical that is generated by the activating enzyme NrdG via chemistry involving S-adenosylmethionine (SAM) and a [4Fe-4S] cluster. The glycyl radical is involved in generation of a transient thiyl (sulfanyl) radical on a cysteine residue, which attacks the substrate, forming a ribonucleotide 3'-radical, followed by water loss to form a ketyl (alpha-oxoalkyl) radical. The ketyl radical gains an electron from a cysteine residue and a proton from formic acid, forming 3'-keto-deoxyribonucleotide and generating a thiosulfuranyl (1lambda4-disulfan-1-yl) radical bridge between methionine and cysteine residues. Oxidation of formate by the thiosulfuranyl radical results in the release of CO2 and regeneration of the thiyl radical. cf. EC 1.17.4.1, ribonucleoside-diphosphate reductase and EC 1.17.4.2, ribonucleoside-triphosphate reductase (thioredoxin).
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + formate
dATP + CO2 + H2O
show the reaction diagram
CTP + formate
dCTP + CO2 + H2O
show the reaction diagram
GTP + formate
dGTP + CO2 + H2O
show the reaction diagram
ribonucleoside 5'-triphosphate + formate
2'-deoxyribonucleoside 5'-triphosphate + CO2 + H2O
show the reaction diagram
TTP + formate
dTTP + CO2 + H2O
show the reaction diagram
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?
UTP + formate
dUTP + CO2 + H2O
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
iron-sulfur centre
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activation of enzyme involves generation of a specific amino acid free radical that is dependent on a reduced Fe-S cluster and S-adenosylmethionine
S-adenosyl-L-methionine
[4Fe-4S]-center
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
iron-sulfur center
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in its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly681)
Zn
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C-terminal region contains a Zn(Cys)4 center. Residues Cys543, Cys546, Cys561, and Cys564 coordinate the metal ion tetrahedrally
Zn2+
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C-terminal region contains a Zn(Cys)4 center
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
bathophenanthroline
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0.02 mM, 97% inhibition
Co2+
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0.02 mM, 75% inhibition
desferal
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0.02 mM, 96% inhibition
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EDTA
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0.1 mM, 96% inhibition
ferrozin
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0.1 mM, 72% inhibition
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dATP
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positive effector for CTP reduction
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4
ATP
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presence of dGTP, pH not specified in the publication, temperature not specified in the publication
0.05 - 0.5
CTP
0.06 - 0.4
GTP
1
UTP
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presence of ATP, pH not specified in the publication, temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012
dATP
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substrate ATP, pH not specified in the publication, temperature not specified in the publication
0.05
dTTP
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substrate GTP, pH not specified in the publication, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.6
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mutant N78A/N311A, pH 8.0, temperature not specified in the publication
5.6
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mutant N311C, pH 8.0, temperature not specified in the publication
10.5
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mutant N78D, pH 8.0, temperature not specified in the publication
11.8
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mutant N78C, pH 8.0, temperature not specified in the publication
87.6
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mutant N78A, pH 8.0, temperature not specified in the publication
207
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mutant N311A, pH 8.0, temperature not specified in the publication
260
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pH 8.5, 22°C
370
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substrate CTP, pH not specified in the publication, temperature not specified in the publication
575
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substrate CTP, presence of ATP, pH not specified in the publication, temperature not specified in the publication
730
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substrate CTP, presence of dATP, pH not specified in the publication, temperature not specified in the publication
1155
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wild-type, pH 8.0, temperature not specified in the publication
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
145000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 84100, calculated, x * 74000 and x * 84000, due to truncation of protein at the site of the glycyl radical, SDS-PAGE
dimer
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2 * 77000, SDS-PAGE
heterotetramer
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2 * 17500, small subunit alpha, plus 2 * 80000, large subunit beta
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
C-terminal region contains a Zn(Cys)4 center structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Residues Cys543, Cys546, Cys561, and Cys564 coordinate the metal ion tetrahedrally
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quantum chemical calculation of reaction mechanism. Carbon dioxide is formed from formate, which is present as a cofactor. The formate first forms a formyl radical. The next step, where the formyl radical protonates the 3'-keto group of the substrate, is rate limiting with a calculated total barrier of 19.9 kcal/mol, in reasonable agreement with the experimental rate-limiting barrier of 17 kcal/mol. Zero-point and entropy effects are quite significant in lowering the barrier
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C260S
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activity comparable to wild-type, mutant is able to undergo truncation at the site of the glycyl radical when the radical-containing enzyme is exposed to oxygen
C453S
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activity comparable to wild-type, mutant is able to undergo truncation at the site of the glycyl radical when the radical-containing enzyme is exposed to oxygen
C543S
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residue is essential for formation of the glycyl radical
C546S
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residue is essential for formation of the glycyl radical
C561S
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residue is essential for formation of the glycyl radical
C564S
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residue is essential for formation of the glycyl radical
C579S
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mutant is able to undergo truncation at the site of the glycyl radical when the radical-containing enzyme is exposed to oxygen
C79S
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residue participates in the actual reduction of the substrate. Mutant is able to undergo truncation at the site of the glycyl radical when the radical-containing enzyme is exposed to oxygen
G580A
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oxygen-dependent cleavage is not possible in this mutant since no radical can be formed at Ala580
N311A
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about 20% of wild-type activtiy
N311C
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less than 1% of wild-type activtiy
N78A
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less than 10% of wild-type activtiy
N78A/N311A
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less than 1% of wild-type activtiy
N78C
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1% of wild-type activtiy
N78D
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1% of wild-type activtiy
C644A
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mutation in a residue of the Zn(Cys)4 center, almost complete loss of activity due to inability to generate the catalytically essential glycyl radical
C647A
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mutation in a residue of the Zn(Cys)4 center, almost complete loss of activity due to inability to generate the catalytically essential glycyl radical
C662A
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mutation in a residue of the Zn(Cys)4 center, almost complete loss of activity due to inability to generate the catalytically essential glycyl radical
C665A
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mutation in a residue of the Zn(Cys)4 center, almost complete loss of activity due to inability to generate the catalytically essential glycyl radical
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine