Information on EC 1.14.13.13 - calcidiol 1-monooxygenase

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

EC NUMBER
COMMENTARY hide
1.14.13.13
-
RECOMMENDED NAME
GeneOntology No.
calcidiol 1-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
calcidiol + NADPH + H+ + O2 = calcitriol + NADP+ + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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redox reaction
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-
-
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reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
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Steroid biosynthesis
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vitamin D3 metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
calcidiol,NADPH:oxygen oxidoreductase (1-hydroxylating)
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CAS REGISTRY NUMBER
COMMENTARY hide
9081-36-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
full-length and truncated enzyme forms
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Manually annotated by BRENDA team
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EMBL
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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Hypercalcaemia in subcutaneous fat necrosis appears to be due to abundant levels of 25-hydroxyvitamin D3-1alpha-hydroxylase in immune infiltrates associated with tissue lesions
physiological function
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mechanisms for involvement of 1alpha-hydroxylase in antimicrobial responses within different barrier tissues, overview
additional information
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25OHD action involves localized extrarenal conversion to 1,25-dihydroxyvitamin D via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase. Expression of 1alpha-hydroxylase is intimately associated with toll-like receptor, TLR, recognition of pathogens in macrophages
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(24R),25-dihydroxycholecalciferol + NADPH + O2
1alpha,24,25-trihydroxycholecalciferol + NADP+ + H2O
show the reaction diagram
(24R),25-dihydroxyvitamin D3 + NADPH + O2
1alpha,24,25-trihydroxyvitamin D3 + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
1alpha-hydroxyvitamin D + NADPH + O2
1alpha,25-dihydroxyvitamin D + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
20-hydroxyvitamin D3 + NADPH + O2
1alpha,20-dihydroxyvitamin D3 + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
23,25-dihydroxycholecalciferol + NADPH + O2
?
show the reaction diagram
24-oxo-23,25-dihydroxycholecalciferol + NADPH + O2
?
show the reaction diagram
24-oxo-25-hydroxycholecalciferol + NADPH + O2
?
show the reaction diagram
25-hydroxycholecalciferol + NADPH + O2
calcitriol + NADP+ + H2O
show the reaction diagram
25-hydroxyvitamin D + NADPH + O2
1alpha,25-dihydroxyvitamin D + NADP+ + H2O
show the reaction diagram
25-hydroxyvitamin D2 + NADPH + O2
1,25-dihydroxyvitamin D2 + NADPH + O2
show the reaction diagram
-
-
-
-
?
25-hydroxyvitamin D3 + NADPH + O2
1,25-dihydroxyvitamin D3 + NADPH + O2
show the reaction diagram
-
-
-
-
?
25-hydroxyvitamin D3 + NADPH + O2
1alpha,25-dihydroxyvitamin D3 + NADP+ + H2O
show the reaction diagram
calcidiol + NADPH + O2
calcitriol + NADP+ + H2O
show the reaction diagram
cholecalciferol + NADPH + O2
25-hydroxyvitamin D3 + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
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
(24R),25-dihydroxycholecalciferol + NADPH + O2
1alpha,24,25-trihydroxycholecalciferol + NADP+ + H2O
show the reaction diagram
25-hydroxycholecalciferol + NADPH + O2
calcitriol + NADP+ + H2O
show the reaction diagram
25-hydroxyvitamin D + NADPH + O2
1alpha,25-dihydroxyvitamin D + NADP+ + H2O
show the reaction diagram
25-hydroxyvitamin D2 + NADPH + O2
1,25-dihydroxyvitamin D2 + NADPH + O2
show the reaction diagram
-
-
-
-
?
25-hydroxyvitamin D3 + NADPH + O2
1,25-dihydroxyvitamin D3 + NADPH + O2
show the reaction diagram
-
-
-
-
?
25-hydroxyvitamin D3 + NADPH + O2
1alpha,25-dihydroxyvitamin D3 + NADP+ + H2O
show the reaction diagram
cholecalciferol + NADPH + O2
25-hydroxyvitamin D3 + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,25-Dihydroxycholecalciferol
22-oxacalcitriol
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vitamin D3 analogue, inhibits in vitro and in vivo
25-hydroxy-3-deoxy-2-oxavitamin D3
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vitamin D3 analogue, competitive inhibition
25-hydroxydihydrotachysterol3
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25-hydroxyvitamin D2
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CYP27B1 shows inhibition when substrate concentrations in the membrane are greater than 4 times Km, more pronounced with 25-hydroxyvitamin D3 than with 25-hydroxyvitamin D2
25-hydroxyvitamin D3
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CYP27B1 shows inhibition when substrate concentrations in the membrane are greater than 4 times Km, more pronounced with 25-hydroxyvitamin D3 than with 25-hydroxyvitamin D2
3-deoxy-2-oxa-9(11)-didehydro-25-hydroxyvitamin D3
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vitamin D3 analogue, competitive inhibition
A-homo-3-deoxy-2-oxa-25-hydroxyvitamin D3
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vitamin D3 analogue, competitive inhibition
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Aminoglutethimide
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enzyme of intact mitochondria
antimycin A
clotrimazole
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in vivo in MCF-7 cells
CN-
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cyanide
Dinitrophenol
diphenyl-p-phenylenediamine
Epidermal growth factor
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decreases mRNA expression in less differentiated cells like Caco-2/AQ and COGA-1A and -1E; i.e. EGF
ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid
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Glutethimide
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ketoconazole
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metapyrone
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Metyrapone
Natural inhibitors
oligomycin
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p-chloromercuribenzoate
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p-Trifluoromethoxyphenylhydrazone
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phosphate
rotenone
Sucrose
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hypertonic
Tris-chloride buffer
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vitamin D3
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,25-Dihydroxycholecalciferol
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increases mRNA expression in highly differentiated cells like Caco-2/15
8-bromo-cAMP
enhances mRNA expression level
adrenodoxin
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mouse and bovine adrenodoxin display similar abilities to support CYP27B1 activity
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CN-
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activates with malate as electron donor
Epidermal growth factor
forskolin
enhances mRNA expression level
malate
succinate
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011
(24R),25-dihydroxycholecalciferol
26
(24R),25-dihydroxyvitamin D3
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pH 7.4, 37C, Michaelis-Menten kinetic parameters
0.00052 - 0.00066
1alpha-hydroxyvitamin D
49
20-hydroxyvitamin D3
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pH 7.4, 37C, parameters using a kinetic model with substrate binding at two inhibitory sites
0.0013
24,25-dihydroxycholecalciferol
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-
0.000088 - 0.89
25-hydroxycholecalciferol
0.00013 - 0.00054
25-hydroxyvitamin D
4.6 - 4.8
25-hydroxyvitamin D2
5.9 - 9.7
25-hydroxyvitamin D3
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.9
(24R),25-dihydroxyvitamin D3
Mus musculus
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pH 7.4, 37C, Michaelis-Menten kinetic parameters
0.0004 - 0.01
1alpha-hydroxyvitamin D
0.14
20-hydroxyvitamin D3
Mus musculus
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pH 7.4, 37C, parameters using a kinetic model with substrate binding at two inhibitory sites
0.0733
25-hydroxycholecalciferol
Rattus norvegicus
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-
0.0008 - 0.385
25-hydroxyvitamin D
0.8 - 0.82
25-hydroxyvitamin D2
0.68 - 0.92
25-hydroxyvitamin D3
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.035
(24R),25-dihydroxyvitamin D3
Mus musculus
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pH 7.4, 37C, Michaelis-Menten kinetic parameters
41298
0.003
20-hydroxyvitamin D3
Mus musculus
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pH 7.4, 37C, parameters using a kinetic model with substrate binding at two inhibitory sites
10258
0.17 - 0.171
25-hydroxyvitamin D2
0.094 - 0.115
25-hydroxyvitamin D3
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.1
Aminoglutethimide
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0.16
metapyrone
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0.001 - 0.002
vitamin D3
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0000001
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female human kidney
0.0000002
0.00000042
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substrate 24-oxo-25-hydroxycholecalciferol, recombinant reconstituted system
0.00000058
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substrate 25-hydroxycholecalciferol, recombinant reconstituted system
0.0000011
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substrate 24-oxo-25-hydroxycholecalciferol
0.0000015
0.0000031
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0.0000043
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substrate 24,25-dihydroxycholecalciferol
0.0000048
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with malate as electron source
0.0000072
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purified cytochrome P-450 component of 1alpha-hydroxylase, reconstituted system
0.0000082
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purified cytochrome P-450 component of 1alpha-hydroxylase, reconstituted system
0.00125
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-
0.0048
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purified enzyme
0.602
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reduction of cytochrome P-450 at non-saturating level of enzyme components
0.687
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ferredoxin activity, reduction of cytochrome c
6.758
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purified ferredoxin, reduction of cytochrome c
11.38
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reconstituted system of cytochrome P-450, cytochrome b5, adrenocortical ferredoxin reductase, and renal ferredoxin
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4 - 8.4
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1 - 7.7
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about 50% of activity maximum at pH 7.1 and pH 7.7
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Manually annotated by BRENDA team
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distributed throughout all brain regions, neurons and glial cells
Manually annotated by BRENDA team
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renal distal tubular epithelial cell line. CYP27B1 gene expression is unchanged after treatement with parathyroid hormone or after high Ca2+ exposure
Manually annotated by BRENDA team
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high enzyme level
Manually annotated by BRENDA team
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expression of 1alpha-OHase is 1.25fold higher in MCF-7 compared to MCF-10A cells. In MCF-10A cells, at least 6 splice variants are detected. Alternative splicing of 1alpha-OHase can regulate the level of active enzyme
Manually annotated by BRENDA team
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renal proximal tubular epithelial cell line. CYP27B1 gene expression is elevated in response to parathyroid hormone. High Ca2+ exposure represses CYP27B1 gene expression in both dose and time-dependent fashion
Manually annotated by BRENDA team
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1-OHase mRNA expression is highest in pre-menopausal women and is increased by all hormones tested. In post-menopausal osteoblasts all hormones except biochainin A and genistein increase 1-OHase mRNA expressions to less extent. In male-derived osteoblasts only dihydrotestosterone and carboxybiochainin A increase 1-OHase mRNA expression
Manually annotated by BRENDA team
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high enzyme and vitamin D receptor expression level, especially in regions of lymphocyte infiltration
Manually annotated by BRENDA team
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prostate cancer cell line, greatly decreased activity of 25-hydroxyvitamin D-1alpha-hydroxylase due to decreased RNA transcription
Manually annotated by BRENDA team
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normal prostate cell line
Manually annotated by BRENDA team
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RT-PCR and immunohistochemic expression analysis
Manually annotated by BRENDA team
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highest activity in proximal tubule
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
-
truncated enzyme form
Manually annotated by BRENDA team
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truncated enzyme form
Manually annotated by BRENDA team
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high enzyme level in large dopaminergic neurons
Manually annotated by BRENDA team
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monocytic cell line
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
11900
-
ferredoxin component, SDS-PAGE
12500
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ferredoxin component, gel filtration
49000
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cytochrome P-450D1alpha, SDS-PAGE
50000
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recombinant enzyme
53000
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MW of ferredoxin component, SDS-PAGE, gel filtration
56000
-
SDS-PAGE
56370
-
predicted from DNA sequence analysis
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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the naturally occuring truncated enzyme form is formed by via proteolytic processing of CYP27A by endogenous protease
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant wild-type Vdh and mutant Vdh-K1 in complex with substrates VD3 and 25(OH)VD3, 20 mg/ml wild-type protein in 20 mM Tris-HCl, pH 7.5, supplemented with 0-0.2% PMbetaCD for VD3 complex or 0-0.2% gammaCD for 25(OH)VD3 complex, 20 mg/ml mutant protein in 20 mM Tris-HCl, pH 7.5, supplemented with 20 mM NaCl, sitting-drop method, 20C. Vdh-WT crystals are grown using reservoir solution containing 0.1 M BisTris, pH 7.5, 50 mM CaCl2, 40-120 mM NaCl or KCl, and 32-40% PEG 400 or PEG 550 monomethyl ether or 20% PEG 1000, Vdh-K1 is crystallized using reservoir solution containing 0.1 M calcium acetate and 10-14% PEG 3350, X-ray diffraction structure determination and analysis at 1.75-3.05 A resolution
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
CYP27B1 is labile during purification despite the presence of 20% glycerol as a stabilizing agent
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-30C, stable without significant loss of activity for at least 6 months
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-80C, in the dark, stable for at least 3 months, cytochrome P-450D1alpha
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0-4C, rapid loss of activity
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4C, prolonged standing of the enzyme, including overnight dialysis, causes marked loss of the holo-enzyme
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
full-length and truncated enzyme forms from liver mitochondria
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partially
-
purification of 1alpha-hydroxylating cytochrome P-450
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purification of cytochrome P-450D1alpha
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purification of ferredoxin component
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recombinant His-tagged CYP27B1 from Escherichia coli strain JM109 by nickel affinity chromatoghraphy, followed by affinity chromatography on a bovine adrenodoxin-coupled resin with elution by 1.0% CHAPS
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recombinant wild-type Vdh and mutant Vdh-K1 from Escherichia coli
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vitamin D3 25- and 1alpha-hydroxylase
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)2D3-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)2D3 synthesis during normal physiology
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expression in COS cells, reconstituted with recombinant cytochrome P-450, recombinant adrenodoxin, and recombinant adrenodoxin reductase
expression in COS cells, vitamin D3 25- and 1alpha-hydroxylase, DNA sequence analysis
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expression in Escherichia coli
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expression in Escherichia coli JM109, coexpression of adrenodoxin and NADPH-adrenodoxin reductase for reconstitution of the system
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expression in LLC-PK1-cells
expression of His-tagged CYP27B1 in Escherichia coli strain JM109
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expression of vitamin D hydroxylases in the VDR-/- mouse model, overview
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expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
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expression of wild-type Vdh and mutant Vdh-K1 in Escherichia coli
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expressionin RAW264.7 cells
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His-tag, expressed in Rhodococcus erythropolis and Escherichia coli
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quantitative PCR analysis of gene expression in different tissues/cells, overview
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
calcitonin induces 25-hydroxyvitamin D3 1alpha-hydroxylase protein levels in AOK-B50 cells transfected with a mouse 25-hydroxyvitamin D3 1alpha-hydroxylase promoter construct (transcription factor C/EBPbeta is involved)
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expression of 1alpha-hydroxylase is induced via toll-like receptor, TLR, recognition of pathogens in macrophages, but not in colonic cells, i.e. Caco-2 and BBe colonic cell lines. Colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D.
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genes CYP2R1 encoding vitamin D 25-hydroxylase, CYP27B1 encoding 25-hydroxyvitamin D-1 alpha hydroxylase and CYP24A1 encoding 1,25-dihydroxyvitamin D(3) 24-hydroxylase are upregulated in clear cell renal cell carcinomas compared with normal tissue. CYP24A1 displays a significantly higher expression in tumors than CYP27B1 and CYP2R1, whereas no differences in the expression of these genes are found in healthy renal tissue
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hyperplastic parathyroid glands from patients with chronic kidney failure normally display a heterogeneous cellularity. 25-hydroxyvitamin D3 1alpha-hydroxylase is expressed at much higher levels in oxyphil cells than in chief cells in these patients. The calcimimetic cinacalcet increases the expression of the enzyme in human parathyroid cultures
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in Klotho knockout mice
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kidney: 25-hydroxycholecalciferol induces a significant downregulation 3 days after injection
kidney: calcitriol and 25-hydroxycholecalciferol induce a significant upregulation 6 days after injection; kidney: parathyroid hormone-related protein induces significant upregulation 6 h after injection
statistically significantly lower protein expression in malignant ovarian tissue
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G125E
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native mutant, no enzyme activity but normal expression level
P382S
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native mutant, no enzyme activity but normal expression level
R107H
-
native mutant, no enzyme activity but normal expression level
R335P
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native mutant, no enzyme activity but normal expression level
Q65A
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site-directed mutagenesis, three-dimensional structure analysis and comparison to the wild-type enzyme
Q65E
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site-directed mutagenesis, three-dimensional structure analysis and comparison to the wild-type enzyme
Q65H
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site-directed mutagenesis, three-dimensional structure analysis and comparison to the wild-type enzyme
Q65L
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site-directed mutagenesis, three-dimensional structure analysis and comparison to the wild-type enzyme
Q65N
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site-directed mutagenesis, three-dimensional structure analysis and comparison to the wild-type enzyme
S408A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, three-dimensional structure analysis and comparison to the wild-type enzyme
S408I
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, three-dimensional structure analysis and comparison to the wild-type enzyme
S408T
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, three-dimensional structure analysis and comparison to the wild-type enzyme
S408V
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, three-dimensional structure analysis and comparison to the wild-type enzyme
T70R/V156L/E216M/E384R
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mutant Vdh-K1, 22fold more active in the hydroxylation of VD3 to 25(OH)VD3 than wild-type Vdh in in vivo bioconversion using Escherichia coli cells. backbone and side chain conformations at the active-site pocket are identical among the substrate-free, VD3-bound, and 25(OH)VD3-bound forms of Vdh-K1, and no induced-fit mechanisms are observed, although VD3 is asymmetric
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine