Information on EC 4.1.99.17 - phosphomethylpyrimidine synthase

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

EC NUMBER
COMMENTARY hide
4.1.99.17
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RECOMMENDED NAME
GeneOntology No.
phosphomethylpyrimidine synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5-amino-1-(5-phospho-D-ribosyl)imidazole + S-adenosyl-L-methionine = 4-amino-2-methyl-5-phosphomethylpyrimidine + 5'-deoxyadenosine + L-methionine + formate + CO
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-amino-2-methyl-5-diphosphomethylpyrimidine biosynthesis
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Metabolic pathways
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Thiamine metabolism
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vitamin B1 metabolism
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SYSTEMATIC NAME
IUBMB Comments
5-amino-1-(5-phospho-D-ribosyl)imidazole formate-lyase (decarboxylating, 4-amino-2-methyl-5-phosphomethylpyrimidine-forming)
Binds a [4Fe-4S] cluster that is coordinated by 3 cysteines and an exchangeable S-adenosyl-L-methionine molecule. The first stage of catalysis is reduction of the S-adenosyl-L-methionine to produce L-methionine and a 5'-deoxyadenosin-5'-yl radical that is crucial for the conversion of the substrate. Part of the pathway for thiamine biosynthesis.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
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-
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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
metabolism
physiological function
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
5-amino-1-(5-phospho-D-ribosyl)imidazole + S-adenosyl-L-methionine
4-amino-2-methyl-5-phosphomethylpyrimidine + 5'-deoxyadenosine + L-methionine + formate + CO
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
5-amino-1-(5-phospho-D-ribosyl)imidazole + S-adenosyl-L-methionine
4-amino-2-methyl-5-phosphomethylpyrimidine + 5'-deoxyadenosine + L-methionine + formate + CO
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S-center
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ThiC carries an oxygen labile [Fe-S] cluster essential for activity
iron-sulfur centre
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S-adenosyl-L-methionine
[4Fe-4S]-center
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4Fe-4S center
binds 1 4Fe-4S cluster per subunit. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine
4Fe-4S cluster
Co2+
a metal ion with octahedral coordination (two strictly conserved histidine residues (H426 and H490) and four water molecules) at the same location as a zinc ion in the bacterial enzyme, and a metal ion with multiple coordinated water molecules in the close vicinity of the substrate binding sites, binding structures, overview
iron-sulfur centre
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-deoxyadenosine
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cooperative inhibition by with Met
adenosine
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uncompetitive versus L-adenosyl-L-methionine
L-homocysteine
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L-methionine
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cooperative inhibition by with 5'-deoxyadenosine
S-adenosyl-L-homocysteine
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competitive versus L-adenosyl-L-methionine
S-methyl-5'-thioadenosine
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additional information
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inhibitiory potencies, overview. No inhibition by product 4-amino-2-methyl-5-phosphomethylpyrimidine, and by cAMP, ADP, ATP, imidazole, aminoimidazole carboxamide ribotides, 5-amino-4-imidazolecarboxylic acid ribotide, CoA, acetyl-CoA, adenine, and 2'-deoxyadenosine
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.017
5-amino-1-(5-phospho-D-ribosyl)imidazole
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pH 8.0, 37°C, recombinant His-tagged enzyme
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Salmonella enterica
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ThiC undergoes ThiC undergoes steady-state turnover, and multiple turnovers with a 5fold molar excess of 5-amino-1-(5-phospho-D-ribosyl)imidazole
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012
5'-deoxyadenosine
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pH 8.0, 37°C, recombinant His-tagged enzyme
0.083
L-methionine
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pH 8.0, 37°C, recombinant His-tagged enzyme
additional information
additional information
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inhibition kinetics, overview
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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strong expression
Manually annotated by BRENDA team
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strong expression
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65451
2 * 65451, recombinant His-tagged DELTAN71-AtTHIC mutant, mass spectrometry and crystal structure
68000
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x * 68000, SDS-PAGE
71994
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x * 71994, calculated from sequence
130000
recombinant His-tagged DELTAN71-AtTHIC mutant, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
2 * 65451, recombinant His-tagged DELTAN71-AtTHIC mutant, mass spectrometry and crystal structure
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged DELTAN71-AtTHIC mutant, by a proteolysis assay (especially alpha-chymotrypsin) coupled with the sitting drop vapor diffusion technique at 18°C, using 0.01 M cobalt (II) chloride hexahydrate, 0.1 M sodium acetate trihydrate, pH 4.6, 1 M 1,6-hexanediol, X-ray diffraction structure determination and analysis at 1.6 A resolution
selenomethionine HMP-P synthase/HMP crystals are grown using the sitting drop vapor diffusion method at 25°C. The crystals are monoclinic, space group P2(1), with unit cell dimension a = 63.3 A, b = 103.4 A, c = 95.4 A and beta = 91.6°
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
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purified recombinant His6-tagged enzyme, 3 min, inactivation
85
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purified recombinant His-tagged wild-type and mutant enzymes, 3 min, inactivation
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
identification of variants of ThiC that have increased sensitivity to oxidative growth conditions
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716133
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged N-terminally truncated mutant DELTAN71-AtTHIC from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21AI by nickel affinity chromatography and gel filtration
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recombinant His6-tagged enzyme from Azotobacter vinelandii, the [4Fe-4S] cluster is reconstituted in vitro prior to freezing of the enzyme at -80°C
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene thiC, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21AI, which also expresses Azotobacter vinelandii [Fe-S] cluster loading genes (iscSUA, hscBA, fdx, orf3, ndK)
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gene thiC, overexpression of His6-tagged enzyme from vector pET-28b(+) in a Azotobacter vinelandii strain overexpressing [Fe-S] cluster-loading genes from plasmid pDB1282
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gene thiC, recombinant expression of His-tagged N-terminally truncated mutant DELTAN71-AtTHIC in Escherichia coli strain BL21(DE3)
heterologous expression of AtTHIC can functionally complement the thiC knock-out mutant of Escherichia coli
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LeThiC under the control of the cauliflower mosaic virus 35S promoter is introduced into the tl mutant by Agrobacterium tumefaciens-mediated transformation. Expression of the wild-type LeThiC gene in the tl mutant is able to complement the mutant to wild type
overexpression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
downregulation of AtTHIC expression by T-DNA insertion at its promoter region results in a drastic reduction of thiamine content in plants and the knock-down mutant thic1 shows albino (white leaves) and lethal phenotypes under the normal culture conditions
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expression of LeThiC is tightly regulated at the transcriptional and posttranscriptional level by multiple factors, such as light, Fe2+ status and thiamine diphosphate-riboswitch. A feedback regulation mechanism is involved in synthesis of the pyrimidine moiety for controlling thiamine synthesis in tomato
the abundance of LeTHIC expression is dependent on light; the expression intensity of LeThic at both the transcriptional (S form mRNA) and protein levels is increased under Fe2+ deficiency (1 or 0 mM Fe2+) compared with Fe2+ sufficiency (10 and 100 mM Fe2+), whereas no effects on LeThiC expression are observed under deficiency of Zn2+ or Mn2+
the THIC gene is negatively regulated by thiamin itself, regulation by conserved regions of mRNA that bind specific metabolites (riboswitches). As the thiamine riboswitch only responds to thiamin diphosphate but not thiamine, the down-regulation of THIC mRNA implies that the externally provided thiamine is converted to thiamine diphosphate inside the cell leading to the conformational change inducing mRNA instability
the transcript level in seedlings grown in 24 h of light is substantially higher than that in seedlings exposed to a single long day cycle (16-h light/8-h dark); THIC transcript is not detectable two days after germination, but is readily detectable at day five
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A527T
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
D468N
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random mutagenesis by by hydroxylamine, inactive mutant
D509G
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
D61N/G513E
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random mutagenesis by by hydroxylamine, inactive mutant
E281K
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
G273N
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
G355D
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random mutagenesis by by hydroxylamine, inactive mutant
G472D
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random mutagenesis by by hydroxylamine, inactive mutant
G479R
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random mutagenesis by by hydroxylamine, inactive mutant
G481S
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random mutagenesis by by hydroxylamine, inactive mutant
G486D
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random mutagenesis by by hydroxylamine, inactive mutant
G92D
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random mutagenesis by by hydroxylamine, inactive mutant
H501Y
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random mutagenesis by by hydroxylamine, inactive mutant
P498L
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
R397H
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random mutagenesis by by hydroxylamine, inactive mutant
R544C
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random mutagenesis by by hydroxylamine, inactive mutant
S247F
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random mutagenesis by by hydroxylamine, inactive mutant
V267M
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
E281K
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random mutagenesis by by hydroxylamine, the mutant shows reduced activity compared to the wild-type enzyme
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G486D
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random mutagenesis by by hydroxylamine, inactive mutant
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G92D
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random mutagenesis by by hydroxylamine, inactive mutant
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S247F
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random mutagenesis by by hydroxylamine, inactive mutant
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additional information
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