Information on EC 2.1.1.107 - uroporphyrinogen-III C-methyltransferase

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

EC NUMBER
COMMENTARY hide
2.1.1.107
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RECOMMENDED NAME
GeneOntology No.
uroporphyrinogen-III C-methyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 S-adenosyl-L-methionine + uroporphyrinogen III = 2 S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
overall reaction
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-
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S-adenosyl-L-methionine + precorrin-1 = S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
(1b)
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-
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S-adenosyl-L-methionine + uroporphyrinogen III = S-adenosyl-L-homocysteine + precorrin-1
show the reaction diagram
(1a)
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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heme metabolism
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Metabolic pathways
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Porphyrin and chlorophyll metabolism
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:uroporphyrinogen-III C-methyltransferase
This enzyme catalyses two sequential methylation reactions, the first forming precorrin-1 and the second leading to the formation of precorrin-2. It is the first of three steps leading to the formation of siroheme from uroporphyrinogen III. The second step involves an NAD+-dependent dehydrogenation to form sirohydrochlorin from precorrin-2 (EC 1.3.1.76, precorrin-2 dehydrogenase) and the third step involves the chelation of Fe2+ to sirohydrochlorin to form siroheme (EC 4.99.1.4, sirohydrochlorin ferrochelatase). In Saccharomyces cerevisiae, the last two steps are carried out by a single bifunctional enzyme, Met8p. In some bacteria, steps 1-3 are catalysed by a single multifunctional protein called CysG, whereas in Bacillus megaterium, three separate enzymes carry out each of the steps, with SirA being responsible for the above reaction. Also involved in the biosynthesis of cobalamin.
CAS REGISTRY NUMBER
COMMENTARY hide
73665-99-3
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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
2 S-adenosyl-L-methionine + uroporphyrinogen III
2 S-adenosyl-L-homocysteine + dihydrosirohydrochlorin + 2 H+
show the reaction diagram
2 S-adenosyl-L-methionine + uroporphyrinogen III
2 S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
S-adenosyl-L-methionine + heptacarboxyporphyrinogen III
?
show the reaction diagram
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-
-
-
?
S-adenosyl-L-methionine + hexacarboxyporphyrinogen III
?
show the reaction diagram
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-
-
-
?
S-adenosyl-L-methionine + pentacarboxyporphyrinogen III
?
show the reaction diagram
-
-
-
-
?
S-adenosyl-L-methionine + precorrin-1
S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + precorrin-2
S-adenosyl-L-homocysteine + trimethylpyrrocorphin
show the reaction diagram
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-
-
-
?
S-adenosyl-L-methionine + uroporphyrinogen I
?
show the reaction diagram
S-adenosyl-L-methionine + uroporphyrinogen III
?
show the reaction diagram
S-adenosyl-L-methionine + uroporphyrinogen III
S-adenosyl-L-homocysteine + precorrin-1
show the reaction diagram
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-
-
?
S-adenosyl-L-methionine + uroporphyrinogen III
S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
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-
-
ir
uroporphyrinogen III + S-adenosyl-L-methionine
precorrin-2 + S-adenosyl-L-homocysteine
show the reaction diagram
additional information
?
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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
2 S-adenosyl-L-methionine + uroporphyrinogen III
2 S-adenosyl-L-homocysteine + dihydrosirohydrochlorin + 2 H+
show the reaction diagram
2 S-adenosyl-L-methionine + uroporphyrinogen III
2 S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
S-adenosyl-L-methionine + precorrin-1
S-adenosyl-L-homocysteine + precorrin-2
show the reaction diagram
D2KI47
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-
-
?
S-adenosyl-L-methionine + precorrin-2
S-adenosyl-L-homocysteine + trimethylpyrrocorphin
show the reaction diagram
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-
-
-
?
S-adenosyl-L-methionine + uroporphyrinogen III
S-adenosyl-L-homocysteine + precorrin-1
show the reaction diagram
D2KI47
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-
-
?
uroporphyrinogen III + S-adenosyl-L-methionine
precorrin-2 + S-adenosyl-L-homocysteine
show the reaction diagram
additional information
?
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uroporphyrinogen III synthase is fused to uroporphyrinogen III methyltransferase in Desulfovibrio vulgaris
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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complete inhibition by 0.1 mM, mechanism unclear
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-homocysteine
uroporphyrinogen III
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0042
heptacarboxyporphyrinogen III
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0.0047
hexacarboxyporphyrinogen III
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0.0034
pentacarboxyporphyrinogen III
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0.0063
S-adenosyl-L-methionine
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0.0004 - 0.052
uroporphyrinogen III
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
38
uroporphyrinogen III
Pseudomonas denitrificans
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00032
S-adenosyl-L-homocysteine
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000044
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recombinant enzyme
0.003
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purified recombinant CobA/HemD
0.0062
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purified enzyme
0.0091
purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at room temperature
PDB
SCOP
CATH
ORGANISM
UNIPROT
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27610
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calculated from DNA sequence
29200
calculated from DNA sequence
39900
calculated from DNA sequence
49930
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calculated from DNA-sequence
50000
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gel filtration
56000
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gel filtration
58200
gel filtration
66000
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gel filtration
67000
SDS-PAGE, uncleaved fusion protein
79000
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dynamic light scattering assay
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
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SDS-PAGE, gel filtration
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using Tris, pH 8.0, 24% (w/v) PEG 6000 for enzyme without substrate, and 0.2 M lithium sulfate, 0.1 M Tris, pH 8.5, 1.26 M ammonium sulfate for enzyme in complex with uroporphyrinogen III
enzyme topology diagramm
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both apoform and with S-adenosyl-L-methionine
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
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thermostable, 17% of activity remaining after 10 min incubation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
complete loss of activity when treated with Triton X-100
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 10 mM Tris, pH 7.7, stable for several weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by NiNTA column
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from recombinant E. coli
from recombinant E. coli using His-tag
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Ni-NTA column chromatography, and gel filtration
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Ni2+-Sepharose column chromatography
recombinant CobA/HemD , HemD, and CobA from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant His-tagged NirE from Escherichia coli by metal chelate chromatography and gel filtration to homogeneity
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recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
50 fold increase of enzyme activity when overexpressed
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complementation experiments with cysteine autotroph strain
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cysG containing plasmid transformed into Escherichia coli CBK103; overexpression of cysG gene
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DNA and amino acid sequence determination and analysis, the uroporphyrinogen III synthase is naturally fused with the methyltransferase, bypassing the need for uroporphyrinogen III decarboxylase activity, overview. Expression of cobA/hemD and isolate hemD or cobA in Escherichia coli strain BL21(DE3)
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EC 2.1.1.107 deficient mutants complemented with cysG from Salmonella typhimurium and Escherichia coli and cobA from Pseudomonas denitrificans, overexpression of yeast gene product in Escherichia coli
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Escherichia coli JM109 used for cloning all recombinant plasmids and expressing the pUC18-derived plasmid, Escherichia coli strain S13009 and BL21(DE3) used for expressing the pQE-derived plasmids and the pET-derived plasmid
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli DH5alpha, BL21(DE3) and RosettaT (DE3) cells
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expressed in Escherichia coli Rosetta (DE3) cells
expression in Escherichia coli
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expression in Escherichia coli and Pseudomonas denitrificans
expression of 5 open reading frames, codon 1120-1959 (cobA) encodes EC 2.1.1.107 activity
gene cobA, expression in Escherichia coli strain JM109(DE3), the recombinant enzyme renders the bacteria morre resistant to potassium tellurite and potassium tellurate
in Escherichia coli CBK103
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in Escherichia coli CBK103, amino acids 202-457 responsible for EC 2.1.1.107 activity
in Escherichia coli JM83 and MV1184
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overexpression of cobA gene in Escherichia coli TB1
overexpression of cysG gene
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recombinant expression of C-terminally His-tagged NirE in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH10B
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D227A
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full activity
D248A
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no transmethylase activity
D303A
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full activity
G224A
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unable to bind S-adenosyl-L-methionine
K270I
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full activity
R298L
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unable to bind S-adenosyl-L-methionine
R309L
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no transmethylase activity
E114Q
the mutant shows strongly reduced activity compared to the wild type enzyme
G189K
the mutant shows increased activity and reduced S-adenosyl-L-methionine-binding ability compared to the wild type enzyme
G189N
the mutant shows increased activity and reduced S-adenosyl-L-methionine-binding ability compared to the wild type enzyme
H161F
the mutant shows reduced activity compared to the wild type enzyme
K102A
the mutant shows no activity and strongly reduced S-adenosyl-L-methionine-binding ability compared to the wild type enzyme
M186L
the mutant shows strongly reduced activity and reduced S-adenosyl-L-methionine-binding ability compared to the wild type enzyme
R111K
the mutant shows strongly reduced activity and reduced S-adenosyl-L-methionine-binding ability compared to the wild type enzyme
R149K
the mutant shows no activity compared to the wild type enzyme
R51K
the mutant shows reduced activity and reduced S-adenosyl-L-methionine-binding ability compared to the wild type enzyme
D47N
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binds about a quarter the quantity of S-adenosyl-L-methionine compared with wild-type, reaction product is only poxidised precorrin-1
F106A
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considerably less soluble than wild-type, no binding of S-adenosyl-L-methionine, no enzymic activity
L49A
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binds about half the quantity of S-adenosyl-L-methionine compared with wild-type, reaction products are oxidised forms of both precorrin-1 and precorrin-2
M184A
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slight enzymic activity
T130A
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considerably less soluble than wild-type, no binding of S-adenosyl-L-methionine
Y183A
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considerably less soluble than wild-type, no binding of S-adenosyl-L-methionine, no enzyminc activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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red fluorescent compounds are associated with the recombinant mature SUMT which are identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis, slightly alter the protein secondary structure
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