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Kirsty J. McLean*1 , Marcus Hans† and Andrew W. Munro* *Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, U.K., and †DSM Biotechnology Center, Delft, The Netherlands Abstract Cholesterol is an essential molecule for eukaryotic life and is an important precursor for a wide range of physiological processes. Biosynthesis and homoeostasis of cholesterol are complex mechanisms that are tightly regulated and interlinked with activities of a number of cytochrome P450 enzymes. These P450s play central critical roles in cholesterol metabolism. Key roles include a rate-limiting reaction in the synthesis of cholesterol itself, and in the oxidative transformations of cholesterol into steroid hormones and bile acids. However, microbial P450s also have important roles that impinge directly on human cholesterol synthesis and oxidation. Recent data reveal that Mycobacterium tuberculosis (which infects more than one-third of the world’s human population) uses P450s to initiate breakdown of host cholesterol as an energy source. Microbial P450s also catalyse industrially important transformations in the synthesis of cholesterol-lowering statin drugs, with clear benefits to humans. The present article reviews the various roles of P450s in human cholesterol metabolism, from endogenous P450s through to microbial oxidases that enable catabolism of human cholesterol, or facilitate production of statins that regulate cholesterol production with positive outcomes in cardiovascular disease. Cholesterol: essential for life Cholesterol is essential for life in mammals (as well as in lower eukaryotes such as yeast and fungi) and has a central role in numerous physiological processes. Cholesterol homoeostasis is vital for brain and central nervous system function [1]. Cholesterol plays a number of critical roles and is the basis of cell membrane structure, regulation and maintenance of membrane fluidity [2]. Cholesterol is a substrate for the synthesis of a number of important biomolecules, in particular steroid hormones, oxysterols, BAs (bile acids) and vitamin D. Cholesterol also has crucial roles in immune system function, gene transcription, enzyme function and protein degradation, and has been implicated in signal transduction and apoptosis [3]. Cholesterol can be obtained from food intake, or synthesized de novo in a pathway that starts with the synthesis of HMG-CoA (3β-hydroxy-3-methylglutaryl-CoA) from acetyl-CoA and acetoacetyl-CoA, catalysed by HMG-CoA synthase. This means that dietary intake and cellular requirement influence cholesterol biosynthesis, and the various processes involved in cholesterol homoeostasis are tightly co-ordinated with complex feedback mechanisms. The brain contains more cholesterol than any other organ, and brain cholesterol is almost all synthesized locally. The myelin is particularly enriched and contains the majority of cholesterol in the central nervous system [4]. Cholesterol Key words: cholesterol, cholesterol homoeostasis, cytochrome P450, statin, steroid, tuberculosis. Abbreviations used: BA, bile acid; CYP, cytochrome P450; FXR, farnesoid X receptor; HMG-CoA, 3β-hydroxy-3-methylglutaryl-CoA. 1 To whom correspondence should be addressed (email [email protected]). Biochem. Soc. Trans. (2012) 40, 587–593; doi:10.1042/BST20120077 has emerging roles in a number of neural diseases such as Alzheimer’s, Huntington’s and Parkinson’s diseases, and multiple sclerosis [5,6]. In membranes, cholesterol is intercalated between phospholipids in the lipid bilayer, reducing acyl chain movement and generating a semi-permeable barrier between cellular compartments, thus contributing to both membrane stability and fluidity. Cholesterol in excess over requirements for complexing cell membrane lipids can move to intracellular membranes, restoring plasma membrane cholesterol levels. This ‘active’ cholesterol is the substrate for production of biomolecules such as vitamin D and can bind to, as well as regulate the function of, many membrane proteins. In addition to its role in membranes, cholesterol is the precursor of steroid hormones where synthesis pathways differ between different species [2]. In mammals, cholesterol is converted, in the process known as steroidogenesis, into pregnenolone (Figure 1) which serves as a precursor to all other natural steroid hormones including progesterone, cortisol, aldosterone and testosterone. Sex steroids are produced from pregnenolone via the intermediate dehydroepiandrosterone, which has a regulatory function and is not considered to be a hormone in its own right [7]. Despite their physiological essentiality, only a small proportion of cholesterol is used in production of steroid hormones. Cholesterol homoeostasis is maintained through elimination pathways and the oxysterols (oxidized derivatives of cholesterol) are important intermediates in steroid and BA synthesis. The additional hydroxy group(s) in the oxysterols increases their polarity and facilitates their flux across the blood–brain barrier and cell membranes, hence oxysterols are often considered the transport form C The Authors Journal compilation C 2012 Biochemical Society Biochemical Society Annual Symposium No. 79 Cholesterol, an essential molecule: diverse roles involving cytochrome P450 enzymes 587 588 Biochemical Society Transactions (2012) Volume 40, part 3 Figure 1 Schematic representation of P450-mediated cholesterol metabolism Major steps involving P450s are highlighted: cholesterol biosynthesis (yellow), BA formation (green) and steroidogenesis (peach). of cholesterol, although other important functional roles are also emerging [8,9]. The major route of cholesterol excretion is through conversion into BAs, which are the end-products of cholesterol utilization. BAs are crucial for cholesterol homoeostasis, where they are required to enable C The C 2012 Biochemical Society Authors Journal compilation dietary lipid solubilization, emulsification and digestion, as well as for the absorption of fat-soluble vitamins [10]. In addition to their role in cholesterol elimination, BAs are natural ligands of nuclear transcription factors such as FXR (farnesoid X receptor) and G-protein-coupled receptors. BAs Biochemical Society Annual Symposium No. 79: Frontiers in Biological Catalysis exert regulatory effects on their own biosynthesis as well as on glucose and lipid metabolism via the activation of FXR and other receptors [11–13]. Catabolism of cholesterol to BAs involves two major pathways generally known as the ‘classic’ (neutral) and the ‘alternative’ (acidic) routes, each utilizing a number (17) of different, mainly oxidative, enzymes [14]. In humans, the neutral pathway leads to synthesis of the primary BAs, chenodeoxycholic and cholic acid, that are often conjugated to amino acids. Primary BA formation begins with modification of the cholesterol steroid ring to 7αhydroxycholesterol by a cytochrome P450 enzyme in the liver (CYP7A1), followed by several enzymatic steps that include further oxidations of both the steroid ring and side chain (Figure 1). In the alternative acid pathway, C27 hydroxylation of the cholesterol side chain precedes sterol ring modification, leading mainly to the formation of chenodeoxycholic acid. Secondary (e.g. deoxycholic and lithocholic acid) and tertiary BAs are converted from primary BAs by anaerobic bacterial modifications within the intestines [15,16]. A minor contribution to BA synthesis occurs in the brain. Here, the (24S)-hydroxylation of cholesterol by the P450 CYP46A1 allows its passage across the blood–brain barrier to further P450-mediated oxidation(s) in the liver [17]. (24S)-Hydroxycholesterol, as well as other oxysterols, activate LXR (liver X receptor) (involved in co-ordinating several cholesterol transport steps) and are potent inhibitors of lipid homoeostasis [18]. P450s play pivotal roles in the complex and highly regulated processes of cholesterol biosynthesis and homoeostasis. Disorders in cholesterol synthesis and metabolism can be genetic or functional, inducing a number of pathogenic processes (e.g. Smith–Lemli–Opitz syndrome, Niemann– Pick type C disease or Antley–Bixler syndrome) that result in mental retardation, developmental malformations or death [19–21]. Cholesterol homoeostasis imbalance is one of the major risk factors associated with cardiovascular disease, the principal cause of death in developed countries. Elevated levels of plasma cholesterol are well established to be involved with increased risk of atherosclerotic diseases and other associated problems, such as strokes. Several classes of therapeutic agents have been developed to treat hypercholesterolaemia. At present, there are five classes of plasma-lipid-lowering drugs on the market: statins, ezetimibe, BA sequestrants, fibrates and nicotinic acids (niacin or vitamin B3 ) [22–24]. Statins are the most commonly prescribed and widely studied agents used in cholesterollowering therapies. Statin use has been shown to have positive effects in the prevention of cardiovascular disease, reducing clinical endpoints in recent efficacy statistics, e.g. a 23% decrease in coronary heart disease or myocardial infarction cases and a 19 % decrease in coronary heart disease mortality [25,26]. Despite the positive aspects of statin therapy and the emergence of numerous effects beyond cholesterol-lowering, adverse statin side effects such as myopathy are often experienced [27]. Development and production of better statin drugs (as well as non-statin alternatives) with reduced side effects are of great interest in the pharmaceutical industry. P450s are important in statin treatment, and human isoforms are known to be induced by statins, and also to metabolize particular statin compounds and to give rise to adverse drug–drug reactions [28,29], showing a link between drug metabolism and cholesterol homoeostasis. P450s have also been shown to have essential roles in industrial production of statins via biotransformation and fermentation procedures [30]. Cytochromes P450 P450s are a large and diverse family of enzymes found in all biological kingdoms. They are cysteine thiolateco-ordinated haem b-containing enzymes and have a characteristic absorption band at 450 nm when the reduced haem iron binds to carbon monoxide. The most common P450 reaction is a mono-oxygenation catalysed following the reductive scission of molecular oxygen bound to the haem iron. Two electrons are required for catalytic function and are usually supplied in two consecutive steps from NAD(P)H via redox partners [31]. There are important differences between eukaryotic P450s (which are frequently involved in xenobiotic bioactivation and detoxification, and in synthesis of important endogenous compounds) and prokaryotic P450s (where roles include participation in pathways for utilization of unusual compounds as carbon sources, antibiotic production, and contributions to pathogen biochemistry). P450s classified into different families share low sequence identity, but still show similarities in overall structural fold and contain a number of highly conserved secondary-structural elements. The variable regions in the P450 structure are those associated with diverse substrate binding and catalysis, and there is also variation to enable distinct redox partner interactions and P450 structural flexibility [32]. As mentioned above, P450s have numerous crucial roles that interplay with cholesterol, ranging from participation in cholesterol synthesis and homoeostasis, BA production, steroid hormone biosynthesis, cholesterol catabolism during mycobacterial infection, and also in the production of cholesterol-lowering drugs. The remainder of the present review discusses a number of these enzymes, with particular focus on the cholesterol-metabolizing P450s that are structurally resolved. P450-mediated cholesterol synthesis: CYP51 CYP51 is the only P450 involved in cholesterol synthesis and is the most evolutionarily conserved enzyme in the CYP superfamily of enzymes. CYP51 (sterol 14α-demethylase) is involved in the first and rate-limiting step in the postsqualene portion of cholesterol biosynthesis. It catalyses three consecutive oxidation reactions involved in removing the 14α-methyl group of sterol precursors, lanosterol (and 24,25-dihydrolanosterol) in humans, as well as enabling the production of human follicular fluid meiosis-activating sterol C The C 2012 Biochemical Society Authors Journal compilation 589 590 Biochemical Society Transactions (2012) Volume 40, part 3 [33]. CYP51 is essential in eukaryotes and is the established target of the azole anti-yeast and anti-fungal drugs. There have also been more recent developments in the use of azoles as anti-parasitic agents targeting the trypanosome CYP51s [34,35]. The structure of human CYP51 was recently solved in the ligand-free and azole inhibitor-bound forms [36]. Human CYP51 has significant differences in both its substrate-access channels and structural flexibility upon ligand binding to CYP51B1 from the pathogen Mycobacterium tuberculosis, the first structurally resolved CYP51 [36]. These differences may facilitate rational design of organism-specific inhibitors, increasing the selectivity of CYP51-targeted drugs. P450-mediated cholesterol homoeostasis CYP46A1 CYP46A1 is one of the four key P450s involved in cholesterol homoeostasis and catalyses the first step in cholesterol elimination from the brain, producing (24S)hydroxycholesterol, which controls cholesterol turnover in the central nervous system. This membrane-permeable form of cholesterol can diffuse naturally across cellular membranes and cross the blood–brain barrier [37]. (24S)Hydroxylation allows transportation of the cholesterol derivative to the liver for the important degradative processes needed for cholesterol homoeostasis, and for its conversion into BAs, or for its conjugation to form glucuronide or sulfate metabolites with regulatory roles [38]. CYP46A1 is expressed in small amounts in the liver and testes and plays important roles in signalling and regulation processes, together with a variety of receptors. There has been controversy as to whether CYP46A1 is directly associated with Alzheimer’s disease, with conflicting results from studies of different populations, although there is evidence accumulating through identification of an increasing number of CYP46A1 clinical polymorphisms [18]. CYP46A1 has broad substrate selectivity aside from cholesterol, with its other substrates encompassing a range of structurally diverse compounds that include other sterols and drugs in clinical practice. The crystal structure of human CYP46A1 was resolved in the substrate-free and substrate-bound forms, as well as for CYP46A1 in complex with four structurally distinct pharmaceutical inhibitor-type molecules [17,37]. An interesting revelation from these studies was the extent of conformational change occurring upon binding different molecules. This pertains to the flexibility and versatility of the P450 structure, and its ability to adapt to the different ligand shapes, while retaining nanomolar ligand-binding affinities as well as integrity of the active-site cavity and wider binding pocket. The functional reaction of CYP46A1 and the structure of CYP46A1 bound to the substrate cholesterol-3sulfate are shown in Figure 2(A). CYP7A1 CYP7A1 catalyses the first step in neutral BA synthesis in the liver, with 7α-hydroxylation of cholesterol being the C The C 2012 Biochemical Society Authors Journal compilation rate-limiting step of this pathway. The formation of 7αhydroxycholesterol is highly regulated by negative feedback through BAs and the rate of primary BA synthesis [39]. CYP7A1 activity is also regulated at the transcriptional level through nuclear receptors responsive to a variety of stimuli such as glucose, insulin, thyroid hormones or diet. CYP7A1 has a critical role in cholesterol homoeostasis, with clinical studies revealing severe disorders of cholesterol and BA metabolism arising from CYP7A1 dysfunction [18]. The structure of human CYP7A1 bound to the sterol 8α,9βcholest-4-en-3-one (by Strushkevich and colleagues at the Structural Genomics Consortium, University of Toronto, Toronto, Canada) is deposited in the PDB, but unpublished to date. The CYP7A1-catalysed conversion of cholesterol into 7α-hydroxycholesterol, and the structure of its complex with 8α,9β-cholest-4-en-3-one are shown in Figure 2(B). CYP27A1 CYP27A1 is expressed in almost all cells of the body and has a major function in the production of 27-hydroxycholesterol, the first step in the acidic BA pathway. It also produces 3βhydroxy-cholestenoic acid, oxygenates BA intermediates in the liver in the neutral pathway, and hydroxylates vitamin D3 in the kidney. 27-Hydroxycholesterol is considered to have diverse roles, for instance in cholesterol elimination from macrophages and arterial endothelial cells, and in immunomodulation [40]. Deficiency in CYP27A1 leads to CTCX (cerebrotendinous xanthomatosis) in humans, a lipid-storage disease with multiple characteristics, including dementia, premature atherosclerosis and retinal abnormalities [41]. No structural data exist for CYP27A1 to date. CYP11A1 CYP11A1 is the mitochondrial cholesterol side-chaincleavage enzyme (P450scc ), catalysing the first step in vertebrate steroid hormone biosynthesis. CYP11A1 is the only enzyme known to convert cholesterol into pregnenolone, the precursor of all steroid hormones. The CYP11A1 reaction occurs in three consecutive steps, with two stereospecific hydroxylations, forming (22R)-hydroxycholesterol and (20R,22R)-dihydroxycholesterol, followed by a C–C bond-cleavage reaction to yield pregnenolone. CYP11A1 has narrow substrate specificity limited to cholesterol, 7dehydrocholesterol and vitamin D3 , although side-chain cleavage has not been observed for vitamin D. The crystal structures of human [42] and bovine [43] CYP11A1 have been solved concurrently by two independent laboratories, both in complexes with substrate (and intermediate) molecules. Comparisons between the structures reveal them to be very similar, with the Cα atom backbones traces aligning with root mean square deviation of 0.95 Å (0.095 nm). The shape and volume of the active-site cavities are also comparable, along with the orientation of the substrate molecules and the majority of the substrate-contact residues. The authors of both papers agree that there are some degrees of both protein and substrate movement and flexibility to facilitate Biochemical Society Annual Symposium No. 79: Frontiers in Biological Catalysis Figure 2 Cholesterol-degradation reactions Schematic representation highlighting the four major human P450s involved in cholesterol oxidation and degradation. In the brain, CYP46A1 produces (24S)-hydroxycholesterol. In (A), the structure of CYP46A1 (PDB code 2Q9F) is shown, with the substrate cholesterol-3-sulfate in magenta sticks and spacefill. In liver, CYP7A1 converts cholesterol into 7α-hydroxycholesterol, the first step in the neutral BA synthesis pathway. In (B), the structure of CYP7A1 (PDB code 3SN5) is shown, with the bound sterol 8α,9β-cholest-4-en-3-one in cyan sticks and spacefill. The ubiquitously expressed CYP27A1 catalyses the formation of 27-hydroxycholesterol, the first step in the acidic BA pathway. There is no known structure for this enzyme. In the steroidogenic tissues, CYP11A1 produces the steroid hormone precursor pregnenolone via three consecutive steps, with the hydroxy intermediates indicated before the final C–C bond-cleavage step. In (C), the structure of CYP11A1 (PDB code 3N9Y) is shown with bound cholesterol in orange sticks and spacefill. In all structures, the secondary structure is represented in grey cartoon, haem is shown in red, iron is shown in orange, and key amino acids involved in substrate binding are shown in green backbone. consecutive reactions in the formation of pregnenolone, although there are differing opinions as to their precise nature. The reaction showing the conversion of cholesterol into pregnenolone, and the structure of human CYP11A1 in complex with cholesterol are shown in Figure 2(C). P450-mediated cholesterol catabolism in Mycobacterium tuberculosis: CYP51B1, CYP125 and CYP142 In contrast with the well-characterized roles involving cholesterol and eukaryotic P450s, it was only recently that important cholesterol-metabolizing functionality was revealed in the intracellular pathogen M. tuberculosis. As in most bacteria, M. tuberculosis does not synthesize sterols, and there was initial surprise when genome sequencing of the M. tuberculosis H37Rv strain revealed the presence of CYP51(B1). CYP51B1 has sterol 14α-demethylase activity, although the enzyme does not appear to have the same critical roles observed in eukaryotes [44]. Thus further studies into the physiology and/or pathogenic roles of the M. tuberculosis CYP51B1 enzyme are needed. Recent studies have demonstrated that cholesterol is essential for M. tuberculosis macrophage entry and that it is the major carbon source for mycobacterial growth during M. tuberculosis infection. Utilization of host (human) cholesterol, particularly during the chronic phase of infection, is now established to be crucial for infection and for M. tuberculosis survival within the host [45]. Furthermore, two key P450 enzymes, CYP125 and CYP142, were demonstrated to be involved in M. tuberculosis host cholesterol catabolism. These P450s perform sequential oxidations of the cholesterol side chain at the C27 position, from the terminal alcohol via an aldehyde and through to the acid. This activity ultimately enables the β-oxidation of the cholesterol side chain [46,47]. CYP125 is the major P450 involved in side-chain oxidation and is located in a gene cluster, the intracellular growth operon (igr), which is important for M. tuberculosis survival in macrophages. CYP142 probably functions as a compensatory enzyme for CYP125 in certain M. tuberculosis strains [48]. These findings highlight the adaptive abilities of this pathogenic bacterium in order for it to survive and maintain essential processes during infection. These data also serve to direct the focus of potential antitubercular compounds towards C The C 2012 Biochemical Society Authors Journal compilation 591 592 Biochemical Society Transactions (2012) Volume 40, part 3 Figure 3 Structure and function of M. tuberculosis CYP125 and CYP142 (A) Schematic reaction of CYP125/CYP142-mediated hydroxylation of cholesterol to 27-hydroxycholesterol, and its subsequent conversion into cholestenoic acid via an aldehyde intermediate. (B) P450 active-site architecture showing the main active-site differences between CYP125 in green, shown with androstenedione (PDB code 3IW1), and CYP142 in yellow (PDB code 2XKR). (C) Structure of CYP125 in complex with cholest-4-en-3-one (magenta spacefill, PDB code 2X5W). The haem is shown in red and iron is shown in orange. Secondary-structural elements are highlighted with α-helices in blue, β-sheets in green, the P450 I-helix in orange and FG-helices in yellow. CYP125 (and CYP142) and the cholesterol-metabolism pathway in M. tuberculosis. CYP125 and CYP142 have both been structurally characterized, and the cholesterol sidechain oxidation reaction(s) performed and the relevant P450 structures are shown in Figure 3. P450-mediated statin synthesis Statins are a class of drugs used to lower ‘bad’ [LDL (lowdensity lipoprotein)] cholesterol levels. They inhibit HMGCoA reductase, the key enzyme catalysing the second (and rate-limiting) step in the cholesterol biosynthesis pathway, resulting in the reduction of HMG-CoA to mevalonate [26]. Statins can be of synthetic origin, or naturally occurring with semi-synthetic derivatives produced. For commercial manufacture, the naturally produced statins are often preferable because of costs associated with chemical synthesis. P450s have been shown to have statin-production roles in strains of filamentous fungi such as Aspergillus terreus, and in the production of lovastatin and pravastatin during fermentation and biotransformation. In lovastatin production, a P450 is essential to convert dihydromonacolin into monacolin J, the precursor to lovastatin [30]. In pravastatin biosynthesis, the precursor molecule compactin is produced naturally in Penicillium citrinum. An essential compactin biotransformation step is performed by the Streptomyces carbophilus P450sca , which catalyses the hydroxylation of compactin to produce pravastatin [49,50]. C The C 2012 Biochemical Society Authors Journal compilation Concluding remarks Cholesterol is a pivotal molecule in human metabolism and its regulation. P450 enzymes play diverse roles in its conversion into steroid hormones and other important molecules. 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Gene 210, 109–116 Received 12 March 2012 doi:10.1042/BST20120077 C The C 2012 Biochemical Society Authors Journal compilation 593