Information on EC 1.14.15.22 - vitamin D 1,25-hydroxylase

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The expected taxonomic range for this enzyme is: Streptomyces griseolus

EC NUMBER
COMMENTARY hide
1.14.15.22
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RECOMMENDED NAME
GeneOntology No.
vitamin D 1,25-hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
calcidiol + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2 = calcitriol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
(2)
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calciol + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ = calcidiol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
(1)
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Steroid biosynthesis
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
calciol,ferredoxin:oxygen oxidoreductase (1,25-hydroxylating)
A P-450 (heme-thiolate) enzyme found in the bacterium Streptomyces griseolus. cf. EC 1.14.14.24, vitamin D 25-hydroxylase and EC 1.14.15.18, calcidiol 1-monooxygenase.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1alpha,25-dihydroxyvitamin D3 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
1alpha,25(R),26-trihydroxyvitamin D3 + 1alpha,25(S),26-trihydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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reaction of mutant R73V/R84A, 25R- and 25S-enantiomers are found in a ratio of 5:1
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?
1alpha-hydroxyvitamin D3 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
1alpha,25-dihydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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-
-
-
?
25-hydroxyvitamin D2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
1alpha,25-dihydroxyvitamin D2 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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reaction of mutants R73A/R84A and R73V/R84A
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?
25-hydroxyvitamin D2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
25,26-dihydroxyvitamin D2 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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reaction of mutants R73A/R84A and R73V/R84A
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?
25-hydroxyvitamin D3 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
1alpha,25-dihydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
-
-
-
-
?
calcidiol + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2
calcitriol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
-
-
-
?
calciol + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+
calcidiol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
-
-
-
?
vitamin D2 + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+
25-hydroxyvitamin D2 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
vitamin D3 + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+
1alpha-hydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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-
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-
?
vitamin D3 + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+
25-hydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O
show the reaction diagram
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-
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-
?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0012
1alpha,25-dihydroxyvitamin D3
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26-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.0065
1alpha-hydroxyvitamin D3
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25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.0022
25-hydroxyvitamin D3
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1alpha-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.00091
calcidiol
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25-hydroxyvitamin D3 1alpha-hydroxylation, pH 7.4, 30°C
0.00054
calciol
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vitamin D3 25-hydroxylation, pH 7.4, 30°C
0.00059
vitamin D2
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vitamin D2 25-hydroxylation, pH 7.4, 30°C
0.0035
vitamin D3
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25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0009
1alpha,25-dihydroxyvitamin D3
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26-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.035
1alpha-hydroxyvitamin D3
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25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.0023
25-hydroxyvitamin D3
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1alpha-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.0023
vitamin D3
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25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.72
1alpha,25-dihydroxyvitamin D3
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26-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
5.5
1alpha-hydroxyvitamin D3
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25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
1.03
25-hydroxyvitamin D3
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1alpha-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
0.67
vitamin D3
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25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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wild-type, 1alpha-hydroxyvitamin D3 hydroxylation activity at C25 2.8 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 2.1 mmol of product per min and mol of protein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
structure of the mutant R73V/R84A with 1alpha,25-dihydroxyvitamin D3, a hydrogen-bond network including the 1alpha-hydroxyl group and several water molecules play an important role in the substrate-binding for 26-hydroxylation
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wild-type to 1.5 A resolution, and mutant R84A, native protein and in complex with 1alpha,25-dihydroxyvitamin D3. The compound is positioned 11 A from the iron atom along the I helix within the pocket. The loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B and F helices, creating a transient binding site
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Streptomyces lividans
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
I243A
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complete loss of activity
I293A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 5.4 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 3.4 mmol of product per min and mol of protein
L180A
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complete loss of activity
R193A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 0.48 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 0.72 mmol of product per min and mol of protein
R193K
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complete loss of activity
R193Q
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 0.33 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 0.31 mmol of product per min and mol of protein
R73A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 30.9 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 20.8 mmol of product per min and mol of protein
R73A/R84A
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variant is also capable of converting vitamin D2 to its active form, that is, 1alpha,25-dihydroxyvitamin D2, via 25-hydroxyvitamin D2, with its 1alpha-hydroxylation activity toward 25-hydroxyvitamin D2 being much lower than that toward 25-hydroxyvitamin D3. The substitutions enhance both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25-hydroxyvitamin D2 from the substrate-binding pocket
R73V/R84A
R84A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 75.7 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 34.5 mmol of product per min and mol of protein
R84F
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mutation gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type
R89A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 0.25 mmol of product per min and mol of protein, no 25-hydroxyvitamin D3 hydroxylation activity at C1alpha
S236A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 4.6 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 3.4 mmol of product per min and mol of protein
V181A
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complete loss of activity
V88A
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1alpha-hydroxyvitamin D3 hydroxylation activity at C25 1.52 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 0.34 mmol of product per min and mol of protein
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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expression of mutant R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter leads to synthesis of 25-hydroxyvitamin D3 and large amounts of 1alpha,25-dihydroxyvitamin D3. In addition, polar metabolites 1alpha,25(R),26-trihydroxyvitamin D3 and 1alpha,25(R),26-trihydroxyvitamin D3 are observed at a ratio of 5:1