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MetaCyc Enzyme: adrenodoxin-NADP+ reductase

Gene: Fdxr Accession Number: G-10915 (MetaCyc)

Species: Rattus norvegicus

Component of:
vitamin D3 24-hydroxylase (summary available)
25-hydroxyvitamin D3 1α-hydroxylase
sterol 26-hydroxylase (summary available)

Summary:
The enzyme has been purified to homogeneity from rat liver mitochondria. An apparent molecular mass of 52 kDa was determined by SDS-PAGE and gel filtration chromatography [Atsuta78].

The partially purified enzyme was active in reconstitution of sterol 26-hydroxylase activity along with a ferredoxin-like iron-sulfur protein and cytochrome P450 27A1 [Atsuta78].

The purified enzyme was active in reconstitution of cytochrome P-450-dependent steroid 26-hydroxylase activity (conversion of cholesterol to 26-hydroxycholesterol) along with NADPH and a ferredoxin. The liver and adrenal mitochondrial ferredoxin reductases and ferredoxins were equally active in the assays. The ferredoxin reductase was also active in reconstitution of NADPH-cytochrome c reductase activity along with adrenal ferredoxin. [Pedersen78a].

Gene Citations: [Sagara99]

Locations: mitochondrial inner membrane

Map Position: [105,342,684 <- 105,351,374]

Molecular Weight of Polypeptide: 54.363 kD (from nucleotide sequence), 52.0 kD (experimental) [Pedersen78a ]

Unification Links: Entrez-gene:79122 , PhosphoSite:P56522 , Pride:P56522 , Protein Model Portal:P56522 , SMR:P56522 , String:10116.ENSRNOP00000004592 , UniProt:P56522

Relationship Links: InterPro:IN-FAMILY:IPR016040 , InterPro:IN-FAMILY:IPR021163

Gene-Reaction Schematic: ?

GO Terms:

Cellular Component: GO:0005743 - mitochondrial inner membrane [Atsuta78]

Credits:
Created 17-Oct-2008 by Fulcher CA , SRI International


Enzymatic reaction of: adrenodoxin-NADP+ reductase

Synonyms: adrenodoxin reductase

EC Number: 1.18.1.6

2 a reduced adrenodoxin + NADP+ <=> 2 an oxidized adrenodoxin + NADPH + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

Reversibility of this reaction is unspecified.

Cofactors or Prosthetic Groups: FAD [Pedersen78a]


Subunit of: vitamin D3 24-hydroxylase

Synonyms: 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial, vitamin D(3) 24-hydroxylase

Species: Rattus norvegicus

Subunit composition of vitamin D3 24-hydroxylase = [Cyp24a1][Fdxr]
         cytochrome P450 24A1 = Cyp24a1 (summary available)
         adrenodoxin-NADP+ reductase = Fdxr (summary available)

Summary:
Using calcidiol as substrate, the Km was determined to be 2.8μM. The enzyme was most active toward calcidiol. The enzyme was inactive toward vitamin D3 and xenobiotics such as benzphetamine [Ohyama91]. The physiological significance of 24-hydroxylated calcidiol has not been firmly established (in [Prosser04]).

Credits:
Created 03-Nov-2008 by Fulcher CA , SRI International


Enzymatic reaction of: 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (vitamin D3 24-hydroxylase)

EC Number: 1.14.13.126

(1S)-1,25-dihydroxy-24-oxocalciol + NADPH + H+ + oxygen <=> (1S)-1,23,25-trihydroxy-24-oxocalciol + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.


Enzymatic reaction of: calcidiol 24-hydroxylase (vitamin D3 24-hydroxylase)

EC Number: 1.14.13.126

calcidiol + NADPH + H+ + oxygen <=> secalciferol + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.


Enzymatic reaction of: calcitriol 24-hydroxylase (vitamin D3 24-hydroxylase)

Synonyms: vitamin D(3) 24-hydroxylase, 25-hydroxyvitamin D3-24-hydroxylase

EC Number: 1.14.13.126

calcitriol + NADPH + H+ + oxygen <=> calcitetrol + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for calcitriol: calcidiol [Ohyama91 ]

Inhibitors (Competitive): 1α-hydroxyvitamin D3 [Ohyama91]

Inhibitors (Unknown Mechanism): ketoconazole [Ohyama91] , carbon monoxide [Ohyama91] , 7,8-benzoflavone [Ohyama91] , vitamin D3 [Ohyama91] , vitamin D2 [Ohyama91]


Subunit of: 25-hydroxyvitamin D3 1α-hydroxylase

Species: Rattus norvegicus

Subunit composition of 25-hydroxyvitamin D3 1α-hydroxylase = [Cyp27b1][Fdxr]
         cytochrome p450 27B1 = Cyp27b1 (summary available)
         adrenodoxin-NADP+ reductase = Fdxr (summary available)

Credits:
Created 31-Oct-2008 by Fulcher CA , SRI International


Enzymatic reaction of: 25-hydroxyvitamin D3 1α-hydroxylase

Synonyms: 25-hydroxyvitamin D-1 alpha hydroxylase, mitochondrial, calcidiol 1-monooxygenase, 25-OHD-1 alpha-hydroxylase, 25-hydroxyvitamin D(3) 1-alpha-hydroxylase, 25-hydroxycholecalciferol 1-hydroxylase, 25-hydroxycholecalciferol 1-monooxygenase

EC Number: 1.14.13.13

calcidiol + NADPH + H+ + oxygen <=> calcitriol + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is physiologically favored in the direction shown.

In Pathways: vitamin D3 biosynthesis

Summary:
Enzyme activity required added adrenodoxin-NADP+ reductase, an oxidized ferredoxin and NADPH [Nakamura97, Eto98]. Assay of the enzyme was pH and buffer-dependent, with Bicine buffer giving high activity [Eto98].

Inhibitors (Unknown Mechanism): metyrapone [Paulson85] , carbon monoxide [Paulson85]

Kinetic Parameters:

Substrate
Km (μM)
Citations
calcidiol
1.8
[Eto98]


Subunit of: sterol 26-hydroxylase

Synonyms: sterol 27-hydroxylase

Species: Rattus norvegicus

Subunit composition of sterol 26-hydroxylase = [Cyp27a1][Fdxr]
         cytochrome P450 27A1 = Cyp27a1 (summary available)
         adrenodoxin-NADP+ reductase = Fdxr (summary available)

Summary:
This enzyme catalyzes the first three sterol side chain oxidations in bile acid biosynthesis via the neutral (classic) pathway (EC 1.14.13.15, EC 1.1.1.161 and EC 1.2.1.40), as well as initiating the alternate (acidic) pathway.

The enzyme also participates in cholesterol homeostasis and vitamin D metabolism ([Usui90] and reviewed in [Russell03, Norlin07]). In rats, this enzyme was found to be subject to negative feedback regulation of transcription by bile acids [Vlahcevic96].

Credits:
Created 17-Oct-2008 by Fulcher CA , SRI International


Enzymatic reaction of: (25R)-3α,7α-dihydroxy-5β-cholestan-26-al dehydrogenase (sterol 26-hydroxylase)

(25R)-3α,7α-dihydroxy-5β-cholestan-26-al + NADPH + oxygen <=> (25R)-3α,7α-dihydroxy-5-β-cholestanate + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: bile acid biosynthesis, neutral pathway


Enzymatic reaction of: (25R)-5β-cholestane-3α,7α,26-triol dehydrogenase (sterol 26-hydroxylase)

(25R)-5β-cholestane-3α,7α,26-triol + NADPH + H+ + oxygen <=> (25R)-3α,7α-dihydroxy-5β-cholestan-26-al + NADP+ + 2 H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: bile acid biosynthesis, neutral pathway


Enzymatic reaction of: 5β-cholestane-3α,7α-diol 27-hydroxylase (sterol 26-hydroxylase)

5β-cholestane-3α,7α-diol + NADPH + H+ + oxygen <=> (25R)-5β-cholestane-3α,7α,26-triol + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: bile acid biosynthesis, neutral pathway


Enzymatic reaction of: (25R)-3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase (sterol 26-hydroxylase)

Synonyms: 3α,7α,12α-trihydroxycholestan-26-al 26 oxidoreductase, cholestanetriol-26-al 26 oxidoreductase, 3α,7α,12α-trihydroxy-5β-cholestan-26-al dehydrogenase, trihydroxydeoxycoprostanal dehydrogenase, THAL-NAD oxidoreductase, 3α,7α,12α-trihydroxy-5β-cholestan-26-al:NAD+ 26-oxidoreductase, (25R)-3α,7α,12α-trihydroxy-5β-cholestan-26-al:NAD+ 26-oxidoreductase

(25R)-3α,7α,12α-trihydroxy-5β-cholestan-26-al + NADPH + oxygen <=> (25R)-3α,7α,12α-trihydroxy-5β-cholestan-26-oate + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: bile acid biosynthesis, neutral pathway

Summary:
This activity (EC 1.2.1.40) was identified in rat liver homogenates prepared by ammonium sulfate fractionation. Radiolabeled reaction products were identified by thin layer chromatography [Masui66]. It was earlier thought to be a separate enzyme, but the activity is now attributed to cytochrome P450 27A1 as shown for the rabbit [Dahlback90] and human [Pikuleva98] enzymes (reviewed in [Russell03, Russell08].


Enzymatic reaction of: (25R)-5β-cholestane-3α,7α,12α,26-tetraol dehydrogenase (sterol 26-hydroxylase)

Synonyms: cholestanetetraol 26-dehydrogenase, 5β-cholestane-3α,7α,12α,26-tetraol dehydrogenase, TEHC-NAD oxidoreductase, 5β-cholestane-3α,7α,12α,26-tetraol:NAD+ 26-oxidoreductase, (25R)-5β-cholestane-3α,7α,12α,26-tetraol:NAD+ 26-oxidoreductase

(25R)-5β-cholestane-3α,7α,12α,26-tetraol + NADPH + oxygen + H+ <=> (25R)-3α,7α,12α-trihydroxy-5β-cholestan-26-al + NADP+ + 2 H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: bile acid biosynthesis, neutral pathway

Summary:
This activity (EC 1.1.1.161) was identified in rat liver homogenates prepared by ammonium sulfate fractionation. Radiolabeled reaction products were identified by thin layer chromatography The aldehyde was also identified by enzymatic and chemical methods [Masui66]. It was earlier thought to be a separate enzyme, but the activity is now attributed to cytochrome P450 27A1 as shown for the rabbit [Dahlback90] and human [Pikuleva98] enzymes (reviewed in [Russell03, Russell08].


Enzymatic reaction of: 5-β-cholestane-3α,7α,12α-triol 27-hydroxylase (sterol 26-hydroxylase)

Synonyms: 5-β-cholestane-3α,7α,12α-triol 26-hydroxylase, sterol 27-hydroxylase, 5-β-cholestane-3α,7α,12α-triol hydroxylase, cholestanetriol 26-hydroxylase, sterol 26-hydroxylase, cholestanetriol 26-monooxygenase, 5β-cholestane-3α,7α,12α-triol,NADPH:oxygen oxidoreductase (26-hydroxylating), C27-steroid 26-hydroxylase

5β-cholestane-3α,7α,12α-triol + NADPH + H+ + oxygen <=> (25R)-5β-cholestane-3α,7α,12α,26-tetraol + NADP+ + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

Alternative Substrates for 5β-cholestane-3α,7α,12α-triol: 5β-cholestane-3α,7α-diol [Okuda88 ] , cholesterol [Okuda88 ]

In Pathways: bile acid biosynthesis, neutral pathway

Summary:
When the purified rat liver enzyme was reconstituted with mitochondrial adrenodoxin and NADPH-adrenodoxin reductase (adrenodoxin-NADP+ reductase) the enzyme hydroxylated 5β-cholestane-3α,7α,12α-triol at position 27. Substitution of microsomal NADPH-cytochrome P450 reductase resulted in loss of activity. Activity toward the other alternative substrates listed here was significantly lower. The enzyme showed no activity toward xenobiotics and was not inhibited by metyrapone [Okuda88].

Inhibitors (Unknown Mechanism): 7,8-benzoflavone [Okuda88] , p-chloromercuribenzoate [Okuda88] , aminoglutethimide [Okuda88] , carbon monoxide [Okuda88]


References

Atsuta78: Atsuta Y, Okuda K (1978). "Isolation of rat liver mitochondrial ferredoxin and its reductase active in the 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 26-hydroxylase." J Biol Chem 253(13);4653-8. PMID: 207706

Dahlback90: Dahlback H, Holmberg I (1990). "Oxidation of 5 beta-cholestane-3 alpha,7 alpha, 12 alpha-triol into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid by cytochrome P-450(26) from rabbit liver mitochondria." Biochem Biophys Res Commun 167(2);391-5. PMID: 2322231

Eto98: Eto TA, Nakamura Y, Taniguchi T, Miyamoto K, Nagatomo J, Maeda Y, Higashi S, Okuda K, Setoguchi T (1998). "Assay of 25-hydroxyvitamin D3 1 alpha-hydroxylase in rat kidney mitochondria." Anal Biochem 258(1);53-8. PMID: 9527847

Masui66: Masui T, Herman R, Staple E (1966). "The oxidation of 5-beta-cholestane-3-alpha, 7-alpha, 12-alpha, 26-tetraol to 5-beta-cholestane-3-alpha, 7-alpha, 12-alpha-triol-26-oic acid via 5-beta-cholestane-3-alpha, 7-alpha, 12-alpha-triol-26-al by rat liver." Biochim Biophys Acta 117(1);266-8. PMID: 5914340

Nakamura97: Nakamura Y, Eto TA, Taniguchi T, Miyamoto K, Nagatomo J, Shiotsuki H, Sueta H, Higashi S, Okuda KI, Setoguchi T (1997). "Purification and characterization of 25-hydroxyvitamin D3 1alpha-hydroxylase from rat kidney mitochondria." FEBS Lett 419(1);45-8. PMID: 9426217

Norlin07: Norlin M, Wikvall K (2007). "Enzymes in the conversion of cholesterol into bile acids." Curr Mol Med 7(2);199-218. PMID: 17346171

Ohyama91: Ohyama Y, Okuda K (1991). "Isolation and characterization of a cytochrome P-450 from rat kidney mitochondria that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3." J Biol Chem 266(14);8690-5. PMID: 2026586

Okuda88: Okuda K, Masumoto O, Ohyama Y (1988). "Purification and characterization of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase from female rat liver mitochondria." J Biol Chem 263(34);18138-42. PMID: 2848026

Paulson85: Paulson SK, DeLuca HF (1985). "Subcellular location and properties of rat renal 25-hydroxyvitamin D3-1 alpha-hydroxylase." J Biol Chem 260(21);11488-92. PMID: 4044567

Pedersen78: Pedersen JI, Saarem K (1978). "Rat liver mitochondrial cytochrome P-450--A C27-steroid 26-hydroxylase." J Steroid Biochem 9(12);1165-8. PMID: 745412

Pedersen78a: Pedersen JI, Godager HK (1978). "Purification of NADPH-ferredoxin reductase from rat liver mitochondria." Biochim Biophys Acta 525(1);28-36. PMID: 687632

Pikuleva98: Pikuleva IA, Babiker A, Waterman MR, Bjorkhem I (1998). "Activities of recombinant human cytochrome P450c27 (CYP27) which produce intermediates of alternative bile acid biosynthetic pathways." J Biol Chem 273(29);18153-60. PMID: 9660774

Prosser04: Prosser DE, Jones G (2004). "Enzymes involved in the activation and inactivation of vitamin D." Trends Biochem Sci 29(12);664-73. PMID: 15544953

Russell03: Russell DW (2003). "The enzymes, regulation, and genetics of bile acid synthesis." Annu Rev Biochem 72;137-74. PMID: 12543708

Russell08: Russell DW (2008). "50 years of advances in bile acid synthesis and metabolism." J Lipid Res. PMID: 18815433

Sagara99: Sagara Y, Watanabe Y, Kodama H, Aramaki H (1999). "cDNA cloning, overproduction and characterization of rat adrenodoxin reductase." Biochim Biophys Acta 1434(2);284-95. PMID: 10525147

Sakaki00: Sakaki T, Sawada N, Komai K, Shiozawa S, Yamada S, Yamamoto K, Ohyama Y, Inouye K (2000). "Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24." Eur J Biochem 267(20);6158-65. PMID: 11012668

Usui90: Usui E, Noshiro M, Ohyama Y, Okuda K (1990). "Unique property of liver mitochondrial P450 to catalyze the two physiologically important reactions involved in both cholesterol catabolism and vitamin D activation." FEBS Lett 274(1-2);175-7. PMID: 2253771

Vlahcevic96: Vlahcevic ZR, Jairath SK, Heuman DM, Stravitz RT, Hylemon PB, Avadhani NG, Pandak WM (1996). "Transcriptional regulation of hepatic sterol 27-hydroxylase by bile acids." Am J Physiol 270(4 Pt 1);G646-52. PMID: 8928794


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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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