Download BiochemicalSociety A nnualSymposium No.79

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
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Biochemical Society Annual Symposium No. 79
Cholesterol, an essential molecule: diverse roles
involving cytochrome P450 enzymes
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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
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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
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[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
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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
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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
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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. Recent
data indicate that the M. tuberculosis pathogen has evolved
to use human cholesterol as an energy source and that an
initial step in this process involves M. tuberculosis P450
enzymes. P450s are also important in the synthesis of statins, a
major class of drugs used to regulate cholesterol levels. P450s
play important roles that affect cholesterol synthesis and its
endogenous and drug-mediated regulation, in steroid and
oxysterol production, and in human cholesterol oxidation
for pathogen energy generation. Thus diverse P450s are
crucial to human cholesterol biochemistry and are important
therapeutic targets.
Funding
We acknowledge the Biotechnology and Biological Sciences
Research Council (BBSRC) and DSM for financial support enabling research on cytochrome P450 systems in relation to
cholesterol metabolism [BBSRC grant numbers BB/G014329/1 and
BB/I019227/1].
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Received 12 March 2012
doi:10.1042/BST20120077
C The
C 2012 Biochemical Society
Authors Journal compilation 593