Information on EC 4.2.3.12 - 6-pyruvoyltetrahydropterin synthase

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

EC NUMBER
COMMENTARY hide
4.2.3.12
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RECOMMENDED NAME
GeneOntology No.
6-pyruvoyltetrahydropterin synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
7,8-dihydroneopterin 3'-triphosphate = 6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cleavage of triphosphate bond
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intramolecular redox reaction
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P-O bond cleavage
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triphosphate elimination
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
drosopterin and aurodrosopterin biosynthesis
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Folate biosynthesis
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Metabolic pathways
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sulfopterin metabolism
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tetrahydrobiopterin biosynthesis I
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tetrahydrobiopterin biosynthesis II
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tetrahydrobiopterin biosynthesis III
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SYSTEMATIC NAME
IUBMB Comments
7,8-dihydroneopterin 3'-triphosphate triphosphate-lyase (6-pyruvoyl-5,6,7,8-tetrahydropterin-forming)
Catalyses triphosphate elimination and an intramolecular redox reaction in the presence of Mg2+. It has been identified in human liver. This enzyme is involved in the de novo synthesis of tetrahydrobiopterin from GTP, with the other enzymes involved being EC 1.1.1.153 (sepiapterin reductase) and EC 3.5.4.16 (GTP cyclohydrolase I) [3].
CAS REGISTRY NUMBER
COMMENTARY hide
171716-27-1
synthase, 6-pyruvoyltetrahydropterin [16-cysteine] (human clone lamda HSY2 gene PCBD subunit) /6-pyruvoyltetrahydropterin synthase [16-cysteine] (human clone lambda HSY2 gene PCBD subunit)
171716-28-2
synthase, 6-pyruvoyltetrahydropterin [25-glutamine] (human clone lambdaHSY2 gene PCBD subunit) /6-pyruvoyltetrahydropterin synthase [25-glutamine] (human clone lambdaHSY2 gene PCBD subunit)
171716-29-3
synthase, 6-pyruvoyltetrahydropterin [de-57-valine] (human clone lambdaHSY2 gene PCBD subunit) /6-pyruvoyltetrahydropterin synthase [de-57-valine] (human clone lambdaHSY2 gene PCBD subunit)
171716-30-6
synthase, 6-pyruvoyltetrahydropterin [87-leucine] (human clone lambdaHSY2 gene PCBD subunit)
97089-82-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
strain AX2
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Manually annotated by BRENDA team
strain AX2
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
transgenic mouse with a replacement of the Pts allele, encoding the 6-pyruvoyl-tetrahydropterin synthase, by an in-frame insertion in exon 2 with the beta-galactosidase gene generating a fusion protein containing the N-terminal 35 amino acids for 6-pyruvoyl-tetrahydropterin synthase followed by the beta-galactosidase
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Manually annotated by BRENDA team
strain PCC 7942
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1'-3H-dihydroneopterin triphosphate
6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
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tritium released is not incorporated in the product
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?
2'-3H-dihydroneopterin triphosphate
6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
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tritium released is not incorporated in the product
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?
6-(L-erythro-1,2-dihydroxypropyl 3-monophosphate)-7,8-dihydropterin
6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin + monophosphate
show the reaction diagram
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the binding constant is 225fold increased with respect to the natural substrate
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?
6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin
6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin
?
show the reaction diagram
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the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP
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?
6-pyruvoyltetrahydropterin
6-carboxy-5,6,7,8-tetrahydropterin + ?
show the reaction diagram
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?
7,8-dihydroneopterin triphosphate
6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
dihydroneopterin triphosphate
6-hydroxymethyldihydropterin + triphosphate + ?
show the reaction diagram
sepiapterin
6-carboxy-5,6,7,8-tetrahydropterin + ?
show the reaction diagram
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?
sepiapterin
7,8-dihydropterin
show the reaction diagram
additional information
?
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the sequence of reaction steps include redox transfer between atoms N5, C6 and C1' and unusual triphosphate elimination at the C2'-C3' bond in the side chain
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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
6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin
6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin
?
show the reaction diagram
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the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP
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-
?
7,8-dihydroneopterin triphosphate
6-pyruvoyl-5,6,7,8-tetrahydropterin + triphosphate
show the reaction diagram
additional information
?
-
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the sequence of reaction steps include redox transfer between atoms N5, C6 and C1' and unusual triphosphate elimination at the C2'-C3' bond in the side chain
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
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required
additional information
-
no cofactor required
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ba2+
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stimulatory effect, 5 mM greatly accelerates activity
Co2+
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free metal enzyme activity reaches 60% of activity of wild type with 0.50 mM and concentrations higher than 0.15 mM completely inactivates the enzyme. Less than 15% of activity with Mg2+ with 5 mM
Sr2+
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5 mM greatly accelerates activity
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-Vinylpyridine
ammonium sulfate
50% inactivation at 400 mM
Ca2+
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10 mM, 40-100% inhibition
Fe2+
slight inhibition
Mg2+
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10 mM, 40-100% inhibition
monoiodoacetate
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N-(7-Dimethyl-amino-4-methyl coumarinyl) maleimide
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Ni2+
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10 mM, 40-100% inhibition
PCMB
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Zn2+
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10 mM, 40-100% inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithioerythritol
EDTA
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1-5 mM, 110-120% activation
additional information
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NADPH is not necessary for activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.8
6-(L-erythro-1,2-dihydroxypropyl 3-monophosphate)-7,8-dihydropterin
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pH 7.4, 37C
0.0022 - 17.7
6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin
0.0005 - 0.164
7,8-dihydroneopterin triphosphate
0.92
sepiapterin
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pH 7.5, 37C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 0.032
6-(L-erythro-1,2-dihydroxypropyl 3-triphosphate)-7,8-dihydropterin
additional information
dihydroneopterin triphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00023
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SSCR activity
0.00034
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SSCR activity
0.00039
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PTPS activity
0.000611
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during the third trimester, enzyme assays at 37C and pH 7.4
0.000714
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during the first trimester, enzyme assays at 37C and pH 7.4
0.000782
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during the second trimester, enzyme assays at 37C and pH 7.4
0.00113
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PTPS activity
0.00891
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PTPS activity
0.01303
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PTPS activity
0.0253 - 0.0255
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pH 7.4, 37C, no differences observed in the enzyme activity between pregnant and non-pregnant women
0.044
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R16C mutant enzyme
0.0574
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0.074
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R25Q mutant enzyme
0.077
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0.09767
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SSCR activity
0.09936
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SSCR activity
0.129
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S19A mutant enzyme
57.4
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additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60 - 80
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3.2fold increase in activity compared to that at 37C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
70% of maximum activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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tissue from normal pregnant women
Manually annotated by BRENDA team
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monkey kidney cells, CRL 1650
Manually annotated by BRENDA team
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larvae
Manually annotated by BRENDA team
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cells from patients with BH4 deficiency
Manually annotated by BRENDA team
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in kidney very weak beta-galactosidase expression in adult and strong expression in newborn mice heterozygous for the Pts-lacZ allele (encoding a fusion protein of N-terminal 35 amino acids of 6-pyruvoyl-tetrahydropterin synthase and beta-galactosidase). Localization in the cytoplasm but not in the nucleus in all cells lining the proximal convoluted tube in adult and newborn mice. No staining in the medulla of newborn mice. Nuclear localization in the thin limb of the Henle loops in adult mice
Manually annotated by BRENDA team
acute lymphoblastic leukemia cell line, 65% homology with rat enzyme
Manually annotated by BRENDA team
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heat instability, it is present in the anterior pituitary endothelial cells but not in posterior pituitary
Manually annotated by BRENDA team
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neuroblastoma cells, CRL 2271
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
72000
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native PAGE
85000 - 87000
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gel filtration
90000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
monomer
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1 * or 2 * 16255, wild-type enzyme occurs as monomeric or dimeric form, electrospray mass spectrometry
pentamer
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5 * 16000, SDS-PAGE; 5 * 16257, electrospray ionization-mass spectrometry
tetramer
trimer
additional information
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each subunit consists of 144 residues with 2497 non-hydrogen atoms
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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no glycoprotein
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no carbohydrates bound
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ePTPS is crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular shaped crystals are obtained. Diffraction data are collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A, and contain six molecules in the asymmetric unit
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X-ray crystallography
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overexpressed as native and selenomethionine-substituted protein and the purified protein is crystallized by the oil-microbatch method at 22C. 2.1 A resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 35.83, b = 95.71, c = 05.65 A. The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 A
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X-ray crystallography, sitting drop vapor diffusion
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X-ray crystal structure of the PTPS homolog from Streptomyces coelicolor, SCO 6650, is solved at 1.5 A resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stable at all steps of purification
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, PIPES 50 mM, stable for 1 year
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-20C and -70C, Tris HCl, several months, stable
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-70C, stable for at least 1 year
-70C, stable for several months
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-70C, stable
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4C, Tris-HCl, 4 weeks, no degradation
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography, gel filtration
affinity chromatography, gel filtration , SDS-PAGE
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affinity chromatography, gel filtration, SDS-PAGE
ammonium sulfate precipitation, Affigel Blue chromatography, DEAE-Bio-gel chromatography, Affigel Blue chromatography, SDS-PAGE
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ammonium sulfate precipitation, DEAE-Sephadex A50 column
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ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, ion exchange, hydroxyapatite chromatography
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ammonium sulfate precipitation, hydroxyapatite column, butyl-toyopearl chromatography, gel filtration, HPLC ion exchange column
ammonium sulfate precipitation, hydroxyapatite column, gel filtration
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ammonium sulfate precipitation, hydroxyapatite column, gel filtration, DEAE-Fractogel 650S column, SDS-PAGE
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extraction, chromatography on a column of Ni-NTA gel
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extraction, chromatography on an amylose resin column
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immobilized metal ion affinity chromatography (Ni2+)
recombinant protein
SDS-PAGE
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using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned in Escherichia coli
cloned in Escherichia coli; expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion protein
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expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion protein
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expressed in Escherichia coli as a His-tagged fusion protein
expressed in Escherichia coli BL21(DE3)
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expression in Cos-1 cells
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expression in Escherichia coli
expression in Escherichia coli and COS-1 cells, wild-type enzyme and 4 naturally occurring mutants
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His-tagged version expressed in Escherichia coli BL21(DE3)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C10A
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50% decrease in activity
D96N
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mutant enzyme that causes PTPS deficiency
Delta1-11
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Flag-tag fused PTPS with N-terminal deletion, 97% decrease in activity
DELTA119-145
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Flag-tag fused PTPS with C-terminal deletion, no detectable activity, no more nuclear staining
DELTA143-145
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Flag-tag fused PTPS with C-terminal deletion, no detectable activity, no more nuclear staining
DELTAV57
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mutant enzyme DELTAV57 is incorrectly folded and thus unstable
K143A
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35% decrease in activity
P87L
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phosphorylated, 30% of activity of wild type enzyme
R25G
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mutant enzyme that causes PTPS deficiency
R87L
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has substantial activity but enhanced sensitivity to local unfolding
T106M
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mutant enzyme that causes PTPS deficiency
V56M
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mutant enzyme that causes PTPS deficiency
V70D
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mutant enzyme that causes PTPS deficiency
C42A
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no catalytic activity, complete loss of metal binding site and activity, it is the only Cys in the active site
E133Q
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1.3% of wild-type activity but similar affinity for the substrate
H23L
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complete loss of metal binding site and activity
H48L
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complete loss of metal binding site and activity
H50L
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complete loss of metal binding site and activity
H89N
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4.3% of wild-type activity but similar affinity for the substrate
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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