Cholesterol 24-hydroxylase

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Cholesterol 24-hydroxylase
Protein CYP46A1 PDB 2Q9F.png
Structure of cholesterol 24-hydroxylase (CYP46A1) in Homo sapiens. Based on PyMOL rendering of PDB 2Q9F ​.
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EC no. 1.14.13.98
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Cholesterol 24-hydroxylase (EC 1.14.13.98), also commonly known as cholesterol 24S-hydroxylase, cholesterol 24-monooxygenase, CYP46, or CYP46A1, is an enzyme that catalyzes the conversion of cholesterol to 24S-hydroxycholesterol. It is responsible for the majority of cholesterol turnover in the human central nervous system. [1] The systematic name of this enzyme class is cholesterol,NADPH:oxygen oxidoreductase (24-hydroxylating).

Contents

This enzyme is a member of the cytochrome P450 (CYP) superfamily of enzymes. Like many other CYP enzymes that act on cholesterol, cholesterol-24 hydroxylase is a monooxygenase that hydroxylates the side-chain of cholesterol. [2]

Because 24S-hydroxycholesterol is more polar than cholesterol, it can more easily pass the blood–brain barrier to exit the brain and pass into the bloodstream, where it can then travel to the liver to be further degraded. [3] [4] This enzyme has also been found at low quantities in the retina, where it performs the same function to a lesser degree. [5]

Genetic cloning of the encoding gene (CYP46A1) was first accomplished in 1999 and an extensive E. coli expression and purification system was later developed in 2003. [2] [6]

Molecular structure

The enzymatic structure of the human cholesterol-24 hydroxylase was determined via crystallography at the Stanford Synchrotron Radiation Lightsource, and was shown to be a 57kDa (500 residue) monomeric heme-containing protein bound to the endoplasmic reticulum in neurons.

Cholesterol-24 hydroxylase active site and heme molecule with bound cholesterol 3-sulfate. Hydrophobic residues interacting with the aliphatic tail of the cholesterol are labelled in white. Created in pyMOL from PDB 2Q9F 24-cholesterol hydroxylase active site with bound cholesterol 3-sulfate.png
Cholesterol-24 hydroxylase active site and heme molecule with bound cholesterol 3-sulfate. Hydrophobic residues interacting with the aliphatic tail of the cholesterol are labelled in white. Created in pyMOL from PDB 2Q9F

Cholesterol-24 hydroxylase is similar in structure to many other cytochrome P450s, possessing, for example, the conserved stretch of 23 hydrophobic residues in the N-terminus that make up a transmembrane-anchoring domain (residues 3-27). [7]

Cholester-24 hydroxylase (CYP46A1) with placement of active site shown in red. created in pyMOL from PDB 2Q9F. Cholesterol-24 hydroxylase (CYP46A1) with active site in red.png
Cholester-24 hydroxylase (CYP46A1) with placement of active site shown in red. created in pyMOL from PDB 2Q9F.

Even so, the cholesterol-24 hydroxylase C-terminus has a unique proline-rich region of 5 repeated proline residues, a structural motif absent in all other related cytochrome p450 enzymes. While the exact function of these proline residues remain highly speculative, it has been shown that the deletion of this region results in a two-fold decrease in the enzyme’s catalytic efficiency. [8]

Binding of cholesterol results in an enzymatic conformational change and a subsequent induced fit of the active site around the cholesterol molecule, anchoring the hydroxylation site (C-24, C-25) near the catalytic center of the enzyme (5.7Å from the iron core of the heme molecule to allow oxyferryl intermediates to perform the cholesterol hydroxylation). A loop region, known as the B'-C loop, has a series of 5 residues (residues 116-120) unique to cholesterol-24 hydroxylase that contribute to the positioning of the cholesterol molecule within the active site. A single cholesterol molecule takes up the entirety of the active site, with the aliphatic tail of the cholesterol held in place by interactions with the following hydrophobic residues: Phe-121, Val-126, Ile-301, Ala-302, Ala-367, Thr-475. The active site is accessed via a single entrance created by two helices (B' and F) and the β1-sheet. [8]

There are no known allosteric regulatory sites.

Enzyme mechanism

Cholesterol-24 hydroxylase mechanism. The heme molecule forms an oxyferryl intermediate that abstracts a hydrogen from cholesterol. The subsequent alkyl intermediate then reacts with the activated oxygen to form the final product. Cholesterol-24 hydroxylase mechanism.png
Cholesterol-24 hydroxylase mechanism. The heme molecule forms an oxyferryl intermediate that abstracts a hydrogen from cholesterol. The subsequent alkyl intermediate then reacts with the activated oxygen to form the final product.

Cholesterol-24 hydroxylase catalyzes the following reaction:

cholesterol + NADPH + H+ + O2 (24S)-24-hydroxycholesterol + NADP+ + H2O

The 4 substrates of this enzyme are cholesterol, NADPH, H+, and O2, and its 3 products are 24S-hydroxycholesterol, NADP+, and H2O. [11]

Like all other cytochrome P450s, cholesterol-24 hydroxylase utilizes an oxyferryl intermediate to hydroxylate cholesterol. The oxyferryl radical takes the hydrogen from carbon-24 to create an alkyl radical intermediate. The cholesterol alkyl radical then combines with the activated oxygen on the heme to create 24S-hydroxycholesterol. [9] [10]

Function

Cholesterol-24 hydroxylase contributes to brain cholesterol homeostasis by hydroxylating cholesterol at carbon-24 to 24S-hydroxycholesterol to allow for elimination of cholesterol from the brain to the liver. [11] Only around 6–7 mg of cholesterol, however, are hydroxylated by this enzyme on a daily basis, suggesting the existence of alternative functions – presently unknown. In vitro experiments have shown that it is also capable of further metabolizing 24S-hydroxycholesterol into 24,25- and 24,27-dihydroxycholesterols. [1]

Cholesterol-24 hydroxylase has a variety of possible substrates, including: elongated steroid chains, cholesterol derivatives, and a variety of drug candidates. [12] [13] As such, it is also likely that it plays a role in lipid metabolism in the brain beyond cholesterol breakdown. Because 24S-hydroxycholesterol (main product of this enzyme) is a major activator of oxysterol liver X receptors (LXR), it is possible that cholesterol-24 hydroxylase may play an indirect regulatory role in the metabolism of lipids in the liver. [2] [14] 24S-hydroxycholesterol also regulates the rate of cholesterol synthesis in the brain, with high levels of 24S-hydroxycholesterol shown to reduce mRNA levels of the following cholesterol synthesis enzymes: HMG CoA reductase, squalene synthase, and FPP synthase. [15]

Clinical significance

Variable expression of cholesterol-24 hydroxylase has been linked to the onset of Alzheimer's disease (AD) in humans. Studies have shown that in AD patients, there is significant decreased expression of cholesterol-24 hydroxylase in neurons. [16] As a result, there is a marked increase of cholesterol in the brain tissue, [17] consistent with the trend observed in AD patients.

Substrates metabolized by cholesterol-24 hydroxylase. The red box contains a list of xenobiotics the enzyme can metabolize. The reactions in blue are commonly observed in the brain as part of maintaining cholesterol homeostasis. Substrates of cholesterol-24 hydroxylase.png
Substrates metabolized by cholesterol-24 hydroxylase. The red box contains a list of xenobiotics the enzyme can metabolize. The reactions in blue are commonly observed in the brain as part of maintaining cholesterol homeostasis.

Neuron degradation in AD has often been attributed to the imbalance in cholesterol homeostasis, and many scientists hypothesize that the lowered expression of cholesterol-24 hydroxylase may be the main cause of this imbalance.

On the other hand, while there is decreased expression in the neurons, there is a contrasting increase of expression in the AD patients' astrocytes, where there is a consequent build-up of the product, 24S-hydroxycholesterol. [18] Recent studies have shown that the increased levels of 24S-hydroxycholesterol in astrocytes may lead to a loss of glial glutamate transporters (EAAT2) and the consequent loss of the glutamate uptake function in the brain, another common symptom observed in AD patients. [19]

Still, the link between expression levels of cholesterol-24 hydroxylase and Alzheimer's Disease remain disputable. While some studies have shown that polymorphisms in the encoding gene for cholesterol-24 hydroxylase have an established positive correlation with AD onset, other publications did not find such an association. [20] [21] Increased expression of cholesterol-24 hydroxylase has also been observed in patients of traumatic brain injury, leading to decreased levels of cholesterol in the plasma membrane. This is hypothesized to be the brain’s typical response to injury. [15]

Cholesterol-24 hydroxylase is easily inhibited by many drugs due to its broad substrate specificity. It has been shown to metabolize bufuralol, progesterone, dextromethorphan, methoxyresorufin, cortisol, diclofenac, phenacetin, and testosterone. [13] The ability for inhibition by various xenobiotics makes this enzyme a prime candidate for drug therapy for AD or other brain injuries.

Related Research Articles

In chemistry, hydroxylation can refer to:

<span class="mw-page-title-main">Cytochrome P450</span> Class of enzymes

Cytochromes P450 are a superfamily of enzymes containing heme as a cofactor that mostly, but not exclusively, function as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various compounds, as well as for hormone synthesis and breakdown. In 1963, Estabrook, Cooper, and Rosenthal described the role of CYP as a catalyst in steroid hormone synthesis and drug metabolism. In plants, these proteins are important for the biosynthesis of defensive compounds, fatty acids, and hormones.

<span class="mw-page-title-main">CYP2E1</span> Protein-coding gene in the species Homo sapiens

Cytochrome P450 2E1 is a member of the cytochrome P450 mixed-function oxidase system, which is involved in the metabolism of xenobiotics in the body. This class of enzymes is divided up into a number of subcategories, including CYP1, CYP2, and CYP3, which as a group are largely responsible for the breakdown of foreign compounds in mammals.

<span class="mw-page-title-main">CYP27A1</span> Protein-coding gene in the species Homo sapiens

CYP27A1 is a gene encoding a cytochrome P450 oxidase, and is commonly known as sterol 27-hydroxylase. This enzyme is located in many different tissues where it is found within the mitochondria. It is most prominently involved in the biosynthesis of bile acids.

<span class="mw-page-title-main">Cholesterol 7 alpha-hydroxylase</span> Protein-coding gene in the species Homo sapiens

Cholesterol 7 alpha-hydroxylase also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1) is an enzyme that in humans is encoded by the CYP7A1 gene which has an important role in cholesterol metabolism. It is a cytochrome P450 enzyme, which belongs to the oxidoreductase class, and converts cholesterol to 7-alpha-hydroxycholesterol, the first and rate limiting step in bile acid synthesis.

<span class="mw-page-title-main">21-Hydroxylase</span> Human enzyme that hydroxylates steroids

Steroid 21-hydroxylase is a protein that in humans is encoded by the CYP21A2 gene. The protein is an enzyme that hydroxylates steroids at the C21 position on the molecule. Naming conventions for enzymes are based on the substrate acted upon and the chemical process performed. Biochemically, this enzyme is involved in the biosynthesis of the adrenal gland hormones aldosterone and cortisol, which are important in blood pressure regulation, sodium homeostasis and blood sugar control. The enzyme converts progesterone and 17α-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively, within metabolic pathways which in humans ultimately lead to aldosterone and cortisol creation—deficiency in the enzyme may cause congenital adrenal hyperplasia.

<span class="mw-page-title-main">Camphor 5-monooxygenase</span>

In enzymology, a camphor 5-monooxygenase (EC 1.14.15.1) is an enzyme that catalyzes the chemical reaction

In enzymology, a cholestanetriol 26-monooxygenase (EC 1.14.13.15) is an enzyme that catalyzes the chemical reaction

In enzymology, a trans-cinnamate 4-monooxygenase (EC 1.14.14.91) is an enzyme that catalyzes the chemical reaction

In enzymology, an unspecific monooxygenase (EC 1.14.14.1) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">CYP4F2</span> Enzyme protein in the species Homo sapiens

Cytochrome P450 4F2 is a protein that in humans is encoded by the CYP4F2 gene. This protein is an enzyme, a type of protein that catalyzes chemical reactions inside cells. This specific enzyme is part of the superfamily of cytochrome P450 (CYP) enzymes, and the encoding gene is part of a cluster of cytochrome P450 genes located on chromosome 19.

<span class="mw-page-title-main">CYP7B1</span> Protein-coding gene in the species Homo sapiens

25-hydroxycholesterol 7-alpha-hydroxylase also known as oxysterol and steroid 7-alpha-hydroxylase is an enzyme that in humans is encoded by the CYP7B1 gene. This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids.

<span class="mw-page-title-main">CYP39A1</span> Protein-coding gene in the species Homo sapiens

CYP39A1 also known as oxysterol 7-α-hydroxylase 2 is a protein that in humans is encoded by the CYP39A1 gene.

<span class="mw-page-title-main">CYP4F11</span> Protein-coding gene in the species Homo sapiens

CYP4F11 is a protein that in humans is encoded by the CYP4F11 gene. This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This gene is part of a cluster of cytochrome P450 genes on chromosome 19. Another member of this family, CYP4F2, is approximately 16 kb away. Alternatively spliced transcript variants encoding the same protein have been found for this gene.

Fatty-acid peroxygenase is an enzyme with systematic name fatty acid:hydroperoxide oxidoreductase (RH-hydroxylating). This enzyme catalyses the following chemical reaction

25-hydroxycholesterol 7alpha-hydroxylase (EC 1.14.13.100, 25-hydroxycholesterol 7alpha-monooxygenase, CYP7B1, CYP7B1 oxysterol 7alpha-hydroxylase) is an enzyme with systematic name cholest-5-ene-3beta,25-diol,NADPH:oxygen oxidoreductase (7alpha-hydroxylating). This enzyme catalyses the following chemical reaction

Cholest-4-en-3-one 26-monooxygenase (EC 1.14.13.141, CYP125, CYP125A1, cholest-4-en-3-one 27-monooxygenase) is an enzyme with systematic name cholest-4-en-3-one,NADH:oxygen oxidoreductase (26-hydroxylating). This enzyme catalyses the following chemical reaction

Cytochrome P450 omega hydroxylases, also termed cytochrome P450 ω-hydroxylases, CYP450 omega hydroxylases, CYP450 ω-hydroxylases, CYP omega hydroxylase, CYP ω-hydroxylases, fatty acid omega hydroxylases, cytochrome P450 monooxygenases, and fatty acid monooxygenases, are a set of cytochrome P450-containing enzymes that catalyze the addition of a hydroxyl residue to a fatty acid substrate. The CYP omega hydroxylases are often referred to as monoxygenases; however, the monooxygenases are CYP450 enzymes that add a hydroxyl group to a wide range of xenobiotic and naturally occurring endobiotic substrates, most of which are not fatty acids. The CYP450 omega hydroxylases are accordingly better viewed as a subset of monooxygenases that have the ability to hydroxylate fatty acids. While once regarded as functioning mainly in the catabolism of dietary fatty acids, the omega oxygenases are now considered critical in the production or break-down of fatty acid-derived mediators which are made by cells and act within their cells of origin as autocrine signaling agents or on nearby cells as paracrine signaling agents to regulate various functions such as blood pressure control and inflammation.

24<i>S</i>-Hydroxycholesterol Chemical compound

24S-Hydroxycholesterol (24S-HC), also known as cholest-5-ene-3,24-diol or cerebrosterol, is an endogenous oxysterol produced by neurons in the brain to maintain cholesterol homeostasis. It was discovered in 1953 by Alberto Ercoli, S. Di Frisco, and Pietro de Ruggieri, who first isolated the molecule in the horse brain and then demonstrated its presence in the human brain.

<span class="mw-page-title-main">25-Hydroxycholesterol</span> Chemical compound

25-Hydroxycholesterol is a derivative of cholesterol, which plays a role in various biological processes in humans and other species. It is involved in cholesterol metabolism, antivirus process, inflammatory and immune response, and survival signaling pathway. 25-hydroxycholesterol is biosynthesized from cholesterol by adding a hydroxyl group at the position 25-carbon of a steroid nucleus. This reaction is catalyzed by cholesterol 25-hydroxylase, a family of enzymes that use oxygen and a di-iron cofactor to catalyze hydroxylation reaction.

References

  1. 1 2 Russell DW, Halford RW, Ramirez DMO, Shah R, Kotti T (2009). "Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain". Annual Review of Biochemistry . 78: 1017–1040. doi:10.1146/annurev.biochem.78.072407.103859. PMC   2837268 . PMID   19489738.
  2. 1 2 3 Pikuleva IA (April 2006). "Cholesterol-metabolizing cytochromes P450". Drug Metabolism and Disposition . 34 (4): 513–520. doi:10.1124/dmd.105.008789. PMID   16434543. S2CID   304953.
  3. ↑ Pikuleva IA (December 2006). "Cytochrome P450s and cholesterol homeostasis". Pharmacology & Therapeutics . 112 (3): 761–773. doi:10.1016/j.pharmthera.2006.05.014. PMID   16872679.
  4. ↑ Russell DW (2003). "The enzymes, regulation, and genetics of bile acid synthesis". Annu. Rev. Biochem. 72: 137–74. doi:10.1146/annurev.biochem.72.121801.161712. PMID   12543708.
  5. ↑ Bretillon L, Diczfalusy U, Bjorkhem I, Maire MA, Martine L, Joffre C, Acar N, Bron A, Creuzot-Garcher C (April 2007). "Cholesterol-24S-hydroxylase (CYP46A1) is specifically expressed in neurons of the neural retina". Current Eye Research . 32 (4): 361–366. doi:10.1080/02713680701231857. PMID   17453958. S2CID   26919085.
  6. ↑ Lund EG, Guileyardo JM, Russell DW (1999). "cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain". Proc. Natl. Acad. Sci. U.S.A. 96 (13): 7238–43. Bibcode:1999PNAS...96.7238L. doi: 10.1073/pnas.96.13.7238 . PMC   22064 . PMID   10377398.
  7. ↑ Mast N, Andersson U, Nakayama K, Bjorkhem I, Pikuleva IA (August 2004). "Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications". Arch. Biochem. Biophys. 428 (1): 99–108. doi:10.1016/j.abb.2004.05.012. PMID   15234274.
  8. 1 2 Mast N, White MN, Bjorkhem I, Johnson EF, Stout CD, Pikuleva IA (July 2008). "Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain". Proceedings of the National Academy of Sciences of the United States of America . 105 (28): 9546–9551. Bibcode:2008PNAS..105.9546M. doi: 10.1073/pnas.0803717105 . PMC   2474539 . PMID   18621681.
  9. 1 2 Munro AW, Girvan HM, McLean KJ (June 2007). "Variations on a (t)heme--novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily". Natural Product Reports . 24 (3): 585–609. doi:10.1039/b604190f. PMID   17534532.
  10. 1 2 Ortiz de Montellano PR (February 2010). "Hydrocarbon hydroxylation by cytochrome P450 enzymes". Chemical Reviews . 110 (2): 932–948. doi:10.1021/cr9002193. PMC   2820140 . PMID   19769330.
  11. 1 2 Lund EG, Xie C, Kotti T, Turley SD, Dietschy JM, Russell DW (2003). "Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover". J. Biol. Chem. 278 (25): 22980–8. doi: 10.1074/jbc.M303415200 . PMID   12686551.
  12. ↑ Pikuleva IA, Norcross R, Andersson U, Shou M, Nakayama K, Bjorkhem I, Pikuleva IA (2003). "Broad substrate specificity of human cytochrome P450 46A1 which initiates cholesterol degradation in the brain". Biochemistry. 42 (48): 14284–92. doi:10.1021/bi035512f. PMID   14640697.
  13. 1 2 Mast N, Charvet C, Pikuleva IA, Stout CD (October 2010). "Structural basis of drug binding to CYP46A1, an enzyme that controls cholesterol turnover in the brain". The Journal of Biological Chemistry . 285 (41): 31783–31795. doi: 10.1074/jbc.M110.143313 . PMC   2951250 . PMID   20667828.
  14. ↑ Shafaati M, Olin M, Bavner A, Pettersson H, Rozell B, Meaney S, Parini P, Bjorkhem I (October 2011). "Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo". Journal of Internal Medicine . 270 (4): 377–387. doi: 10.1111/j.1365-2796.2011.02389.x . PMID   21486371.
  15. 1 2 Cartagena CM, Burns MP, Rebeck GW (March 2010). "24S-hydroxycholesterol effects on lipid metabolism genes are modeled in traumatic brain injury". Brain Research . 1319: 1–12. doi:10.1016/j.brainres.2009.12.080. PMC   2826556 . PMID   20053345.
  16. ↑ Winblad B, Russell DW, Bjorkhem I (2001). "On the turnover of brain cholesterol in patients with Alzheimer's disease. Abnormal induction of the cholesterol-catabolic enzyme CYP46 in glial cells". Neurosci. Lett. 314 (1–2): 45–8. doi:10.1016/S0304-3940(01)02277-7. PMID   11698143. S2CID   37449539.
  17. ↑ Desai P, DeKosky ST, Kamboh MI (August 2002). "Genetic variation in the cholesterol 24-hydroxylase (CYP46) gene and the risk of Alzheimer's disease". Neuroscience Letters . 328 (1): 9–12. doi:10.1016/s0304-3940(02)00443-3. PMID   12123847. S2CID   24909348.
  18. ↑ Brown J, Theisler C, Silberman S, Magnuson D, Gottardi-Littell N, Lee JM, Yager D, Crowley J, Sambamurti K, Rahman MM, Reiss AB, Eckman CB, Wolozin B (August 2004). "Differential expression of cholesterol hydroxylases in Alzheimer's disease". The Journal of Biological Chemistry . 279 (33): 34674–34681. doi: 10.1074/jbc.M402324200 . PMID   15148325.
  19. ↑ Tian G, Kong Q, Lai L, Ray-Chaudhury A, Lin CLG (May 2010). "Increased expression of cholesterol 24S-hydroxylase results in disruption of glial glutamate transporter EAAT2 association with lipid rafts: a potential role in Alzheimer's disease". Journal of Neurochemistry . 113 (4): 978–989. doi:10.1111/j.1471-4159.2010.06661.x. PMC   3010752 . PMID   20193040.
  20. ↑ Kolsch H, Lutjohann D, Ludwig M, Schulte A, Ptok U, Jessen F, von Bergmann K, Rao ML, Maier W, Heun R (2002). "Polymorphism in the cholesterol 24S-hydroxylase gene is associated with Alzheimer's disease". Molecular Psychiatry . 7 (8): 899–902. doi: 10.1038/sj.mp.4001109 . PMID   12232784.
  21. ↑ Ma SL, Tang NLS, Lam LCW, Chiu HFK (March 2006). "Polymorphisms of the cholesterol 24-hydroxylase (CYP46A1) gene and the risk of Alzheimer's disease in a Chinese population". International Psychogeriatrics / IPA . 18 (1): 37–45. doi:10.1017/S1041610205003108. PMID   16734927. S2CID   29923846.
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