Information on EC 4.2.1.96 - 4a-hydroxytetrahydrobiopterin dehydratase

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

EC NUMBER
COMMENTARY hide
4.2.1.96
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RECOMMENDED NAME
GeneOntology No.
4a-hydroxytetrahydrobiopterin dehydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4a-hydroxypterin = (6R)-6-(L-erythro-1,2-dihydroxypropyl)-7,8-dihydro-6H-pterin + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination of H2O
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
L-phenylalanine degradation I (aerobic)
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L-phenylalanine degradation V
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phenylalanine metabolism
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SYSTEMATIC NAME
IUBMB Comments
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4a-hydroxypterin hydro-lyase [(6R)-6-(L-erythro-1,2-dihydroxypropyl)-7,8-dihydro-6H-pterin-forming]
Catalyses the dehydration of 4a-hydroxytetrahydrobiopterins
CAS REGISTRY NUMBER
COMMENTARY hide
204788-56-7
pterin-4a-carbinolamine dehydratase (Aquifex aeolicus gene phhB), genBank AE000671-derived protein GI 2982796
87683-70-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
DcoH2 (dimerization cofactor of HNF1alpha)
Uniprot
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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PCD deficiency causes in newborns a mild form of hyperphenylalaninaemia with persistent high urinary levels of primapterin. Affected patients appear completely normal, but have elevated phenylalanine levels at birth, which normalize after few months of life and remain normal or just above the normal range of phenylalanine with an unrestricted diet
metabolism
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the PCD activity of the homoteterameric enzyme is involved in the aromatic amino acid 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
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4a-hydroxypterin
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-7,8-dihydro-6H-pterin + H2O
show the reaction diagram
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-
-
?
(6R)-6-(L-erythro-1,2-dihydroyxypropyl)-5,6,7,8-tetrahydro-4a-hydoxypterin
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-7,8-dihydro-6H-pterin + H2O
show the reaction diagram
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-
-
?
4a-Hydroxy-6(R)-dihydroxypropyltetrahydropterin
?
show the reaction diagram
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-
-
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4a-Hydroxy-6(S)-methyltetrahydropterin
Quinoid 6(S)-methyldihydropterin
show the reaction diagram
4a-Hydroxy-6(S)-propyltetrahydropterin
Quinoid 6(S)-propyldihydropterin
show the reaction diagram
4a-hydroxy-tetrahydrobiopterin
7,8-dihydrobiopterin + H2O
show the reaction diagram
4a-hydroxy-tetrahydropterin
7,8-dihydropterin + H2O
show the reaction diagram
4a-hydroxytetrahydrobiopterin
?
show the reaction diagram
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4alpha-hydroxy-6(S)-methyltetrahydropterin
?
show the reaction diagram
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-
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-
?
6(S)-methyl-4a-hydroxy-tetrahydropterin
6(S)-methyl-7,8-dihydropterin + H2O
show the reaction diagram
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-
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6,6-dimethyl-4a-hydroxy-tetrahydropterin
6,6-dimethyl-7,8-dihydropterin + H2O
show the reaction diagram
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pterin-4alpha-carbinolamine
quininoid dihydropterin + H2O
show the reaction diagram
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?
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
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
contains no metals
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
diethyldicarbonate
inactivation is complete when about 1.2 His per subunit are derivatized
glycerol
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dietary glycerol leads to a small reduction in the mRNA level of the enzyme PCD/DCoH only in the liver, not in kidney, 40% glycerol diet lead to 2.5fold reduced activity in liver cytosol
Quinoid 6(S)-methyldihydropterin
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Quinoid 6(S)-propyldihydropterin
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glycerol
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leads to a slight increase in enzyme activity only in the kidney, not in liver, when feeded as a 40% glycerol diet
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004 - 0.0053
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4a-hydroxypterin
0.006 - 390
4a(R)-hydroxy-6(S)-methyltetrahydropterin
0.0015
4a(R)-hydroxy-6(S)-propyltetrahydropterin
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pH 8.4 or pH 7.4
0.0015 - 0.03
4a(S)-hydroxy-6(R)-methyltetrahydropterin
0.0025 - 0.003
4a-Hydroxy-6(S)-methyltetrahydropterin
0.0013
4a-Hydroxy-6(S)-propyltetrahydropterin
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fetal and adult enzyme
0.0047 - 0.0052
4a-hydroxytetrahydrobiopterin
0.007 - 2.2
6(S)-methyl-7,8-dihydropterin-4a-carbinolamine
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
23 - 24
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydro-4a-hydroxypterin
8.6 - 10
4a(R)-hydroxy-6(S)-methyltetrahydropterin
2.4 - 10
4a(S)-hydroxy-6(R)-methyltetrahydropterin
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0044
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renal cytosol
0.0134
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hepatic cytosol
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
11000
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4 * 11000, SDS-PAGE
11800
4 * 11800, mutant enzyme H61A, H62A and H79A, electrospray mass spectrometry
11867
4 * 11867, wild type enzyme, electrospray mass spectrometry
12675
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4 * 12675, mutant enzyme C81S, electrospray-ionisation mass spectrometry
12690
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4 * 12690, wild type enzyme, electrospray-ionisation mass spectrometry
12744
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4 * 12744, mutant C81R, electrospray-ionisation mass spectrometry
24200
asymmetric unit contains two subunits 2 * 24200
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion method at 4°C, 1.6 A resolution crystal structure, space group P3(1)21, unit cell length: a = b = 57.92 A, c = 114.63 A
purified recombinnat detagged mutant T51S enzyme, 13 mg/ml protein from 0.1 M HEPES-Na, pH 7.5, 2% v/v PEG 400, and 2.0 M ammonium sulfate, X-ray diffraction structure determination and analysis at 1.8 A resolution
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recombinant enzyme
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sitting drop vapour diffusion, 2V6S: 1.4 M ammonium sulfate, 1 M lithium sulfate, 2 mM 7,8-dihydrobiopterin, pH 7.6, space group P 43 21 2, resolution 2.22 A, complex with substrate-like ligand 7,8-dihydrobiopterin TgPCD:BH2 complex 2V6T: 0.4 M potassium-sodium tartrate, pH 7.6, space group P 43 21 2, resolution 3.1 A, apo-enzyme 2V6U: 1.4 M ammonium sulfate, 1 M lithium sulfate, pH 7.6, space group P 43 21 2, resolution 1.6 A
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
no decrease in activity upon dilution and storage at 4°C
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, stable
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mutant proteins require the inclusion of 10% glycerol in the storage buffer to maintain solubility
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by affinity chromatography over a HisTrap nickel chelating column, followed by TEV cleavage/dialysis at 4°C overnight and then further passage over the a HisTrap nickel column to remove the tag, contaminants and the His-tagged TEV protease. A final purification step using a Superdex 75 size-exclusion column is performed
DcoH2 (dimerization cofactor of HNF1alpha)
recombinant
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recombinant enzyme
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recombinant GST-tagged enzyme from Escherichia coli by glutathione affinity chromatography, tag cleavage by thrombin, and p-aminobenzamidine affinity chromatography
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wild type and mutant enzymes Cys81Ser and Cys81Arg
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wild type and mutant enzymes E69A, D100N, H73A, H74A, H91A, W81A, and G72S and double-mutant H73A,H74A expressed in Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli as GST-tagged enzyme
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expression in Escherichia coli BL21 (DE3). There is a high degree of structural similarity between Toxoplasma gondii PCD and the mammalian orthologue, Rattus norvegicus PCD, which is identical to the human enzyme
expression in Escherichia coli BL21(DE3)pLysS
expression in recombinant Escherichia coli strain BL21(DE3) tnaA-, co-expression with Chromobacterium violaceum L-phenylalanine 4-hydroxylase mutant L101Y-W180F, Escherichia coli dihydropteridine reductase, and Bacillus subtilis glucose dehydrogenase genes for installation of 5-hydroxy-L-tryptophan biosynthesis in the recombinant Escherichia coli strain, method optimization and development of a cofactor regeneration system for L-tryptophan hydroxylation for enhanced synthesis of 5-hydroxy-L-tryptophan
overexpression in Escherichia coli
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plasmids pJS11, which carries phhA, and pBluescript, carrying phhB transformed simultaneously by electroporation into Escherichia coli strains JP2255, JP2255 folM, and JP2255 folX
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targeting of full-length GFP-tagged PCD sequence to chloroplasts in Arabidopsis thaliana mesophyll protoplasts
the 18 amino acid-truncated mutant E87T cannot be overexpressed in Escherichia coli; wild type and mutant enzyme C82R overexpressed in Escherichia coli
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wild-type and mutant enzymes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C81R
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mutant enzyme Cys81Arg has significantly lower activity
E57A
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the His79Ala mutant and the His79Ser mutant exhibit about 40% the activity of the wild-type enzyme. In the mutant enzymes His61Ala and His62Ala the activity is reduced to 10%. In the mutant enzymes Asp60Ala and Arg87Ala the activity is reduced to 30%. The Glu57Ala mutant and the His61Ala,His62Ala double mutant show no activity
E97K
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a biopsy of duodenal mucosa from a patient with homozygous E97K mutation has 17% of normal activity
H61A
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the His79Ala mutant and the His79Ser mutant exhibit about 40% the activity of the wild-type enzyme. In the mutant enzymes His61Ala and His62Ala the activity is reduced to 10%. In the mutant enzymes Asp60Ala and Arg87Ala the activity is reduced to 30%. The Glu57Ala mutant and the His61Ala,His62Ala double mutant show no activity
H61A/H62A
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the His79Ala mutant and the His79Ser mutant exhibit about 40% the activity of the wild-type enzyme. In the mutant enzymes His61Ala and His62Ala the activity is reduced to 10%. In the mutant enzymes Asp60Ala and Arg87Ala the activity is reduced to 30%. The Glu57Ala mutant and the His61Ala,His62Ala double mutant show no activity
H62A
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the His79Ala mutant and the His79Ser mutant exhibit about 40% the activity of the wild-type enzyme. In the mutant enzymes His61Ala and His62Ala the activity is reduced to 10%. In the mutant enzymes Asp60Ala and Arg87Ala the activity is reduced to 30%. The Glu57Ala mutant and the His61Ala,His62Ala double mutant show no activity
H79A
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the His79Ala mutant and the His79Ser mutant exhibit about 40% the activity of the wild-type enzyme. In the mutant enzymes His61Ala and His62Ala the activity is reduced to 10%. In the mutant enzymes Asp60Ala and Arg87Ala the activity is reduced to 30%. The Glu57Ala mutant and the His61Ala,His62Ala double mutant show no activity
H79S
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the His79Ala mutant and the His79Ser mutant exhibit about 40% the activity of the wild-type enzyme. In the mutant enzymes His61Ala and His62Ala the activity is reduced to 10%. In the mutant enzymes Asp60Ala and Arg87Ala the activity is reduced to 30%. The Glu57Ala mutant and the His61Ala,His62Ala double mutant show no activity
N61D
The decreased ability of the N61D mutant to affect HNF1alpha-dependent DNA binding is likely a direct result of altered quaternary structure.
N61D/Q45R/K98Q
site-directed mutagenesis, triple DCoHa mutant (Q45R/K98Q/N61D) is unable to affect HNF1alpha-dependent DNA binding in vitro.
Q45R/K98Q
mutant Q45R/K98Q is not able to affect HNF1alpha-dependent DNA binding in vitro
D100N
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the single mutants H73A and H74A and the double mutant H73A,H74A are completely inactive. The activity of the mutant enzymes H91A and E69A is 40% of the activity of the wild type enzyme. The activity of the mutant enzymes D100N is 10% of the activity of the wild type enzyme
E69A
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the single mutants H73A and H74A and the double mutant H73A,H74A are completely inactive. The activity of the mutant enzymes H91A and E69A is 40% of the activity of the wild type enzyme. The activity of the mutant enzymes D100N is 10% of the activity of the wild type enzyme
H73A
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the single mutants H73A and H74A and the double mutant H73A,H74A are completely inactive. The activity of the mutant enzymes H91A and E69A is 40% of the activity of the wild type enzyme. The activity of the mutant enzymes D100N is 10% of the activity of the wild type enzyme
H73A/H74A
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the single mutants H73A and H74A and the double mutant H73A,H74A are completely inactive. The activity of the mutant enzymes H91A and E69A is 40% of the activity of the wild type enzyme. The activity of the mutant enzymes D100N is 10% of the activity of the wild type enzyme
H74A
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the single mutants H73A and H74A and the double mutant H73A,H74A are completely inactive. The activity of the mutant enzymes H91A and E69A is 40% of the activity of the wild type enzyme. The activity of the mutant enzymes D100N is 10% of the activity of the wild type enzyme
H91A
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the single mutants H73A and H74A and the double mutant H73A,H74A are completely inactive. The activity of the mutant enzymes H91A and E69A is 40% of the activity of the wild type enzyme. The activity of the mutant enzymes D100N is 10% of the activity of the wild type enzyme
H61A
mutant enzyme H61A shows no dehydratase activity with 4a(R)-hydroxy-6(R)-methyltetrahydropterin. Mutant enzyme H79A shows no dehydratase activity with 4a(S)-hydroxy-6(R)-methyltetrahydropterin. The Km-value for 4a(S)-hydroxy-6(R)-methyltetrahydropterin is comparable to the Km-value of the wild type enzyme. The turnover number of the mutant enzyme H62A is 24% of that with the wild type enzyme for the 4a(R),6(S)-isomer and the 4a(S),6(R)-isomer
T51S
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the point mutation at the enzyme tetramer interface overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1alpha. Presence of an ordered water molecule at the tetramer interface, which may destabilize the homotetramer
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DCoH2 unfolding through guanidine is reversible, kinetics, equilibrium unfolding data fit to a two-state model with no apparent intermediate, but folding intermediates are detectable. Proposal of an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly
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