new per ox i some complete
TRANSCRIPT
-
7/30/2019 New Per Ox i Some Complete
1/41
Biochemistry of MammalianPeroxisomes Revisited
Ronald J.A. Wanders and Hans R. Waterham
Department of Clinical Chemistry and Pediatrics, Laboratory Genetic MetabolicDisease, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam
The Netherlands; email: [email protected], [email protected]
Annu. Rev. Biochem.2006. 75:295332
First published online as aReview in Advance on
March 22, 2006
The Annual Review of
Biochemistry is online atbiochem.annualreviews.org
doi: 10.1146/annurev.biochem.74.082803.133329
Copyright c 2006 byAnnual Reviews. All rightsreserved
0066-4154/06/0707-0295$20.00
Key Words
fatty acid oxidation, plasmalogens, reactive oxygen species, genediseases
Abstract
In this review, we describe the current state of knowledge abthe biochemistry of mammalian peroxisomes, especially human poxisomes. The identification and characterization of yeast muta
defective either in the biogenesis of peroxisomes or in one of
metabolic functions, notably fatty acid beta-oxidation, combinwith the recognition of a group of genetic diseases in man, wher
these processes are also defective, have provided new insightsall aspects of peroxisomes. As a result of these and other stud
the indispensable role of peroxisomes in multiple metabolic paways has been clarified, and many of the enzymes involved in th
pathways have been characterized, purified, and cloned. One asp
of peroxisomes, which has remained ill defined, is the transportmetabolites across the peroxisomal membrane. Although it is cl
that mammalian peroxisomes under in vivo conditions are clostructures, which require the active presence of metabolite tran
porter proteins, much remains to be learned about the permeabiproperties of mammalian peroxisomes and the role of the four h
ATP-binding cassette (ABC) transporters therein.
295
-
7/30/2019 New Per Ox i Some Complete
2/41
Contents
INTRODUCTION.. . . . . . . . . . . . . . . . 296
PEROXISOMAL PROTEINS . . . . . . 297When to Call a Protein a
Peroxisomal Protein? . . . . . . . . . . 297
Strategies to Identify Putative
Peroxisomal Proteins . . . . . . . . . . 300Strategies to Demonstrate the
Peroxisomal Localization of
Proteins . . . . . . . . . . . . . . . . . . . . . . 300SELECTED METABOLIC
PATHWAYS . . . . . . . . . . . . . . . . . . . . . 301
Oxygen Metabolism, ReactiveOxygen Species, and Reactive
Nitrogen Species Metabolism . . 301Ether-Phospholipid Biosynthesis . 302
Peroxisomal Fatty Acid
Beta-Oxidation... . . . . . . . . . . . . . 305Peroxisomal Fatty Acid
Alpha-Oxidation . . . . . . . . . . . . . . 311Glyoxylate Metabolism . . . . . . . . . . . 312
Amino Acid Catabolism. . . . . . . . . . . 313Pentose Phosphate Pathway. . . . . . . 313
Polyamine Oxidation . . . . . . . . . . . . . 313Miscellaneous Peroxisomal
Enzyme Activities . . . . . . . . . . . . . 314
Isoprenoid and CholesterolMetabolism . . . . . . . . . . . . . . . . . . . 314
BIOCHEMISTRY OF HUMANPEROXISOMAL DISORDERS. . 315
MOUSE MODELS FOR
PEROXISOMAL DISORDERS. . 316PEROXISOMAL METABOLITE
TRANSPORT . . . . . . . . . . . . . . . . . . 319Permeability Properties of
Peroxisomes . . . . . . . . . . . . . . . . . . 319The Intraperoxisomal pH . . . . . . . . . 322
Peroxisomal ABC Transporters. . . . 322
Peroxisomal ATP Transporter . . . . . 324Other Putative Peroxisomal
Transporters . . . . . . . . . . . . . . . . . . 325CONCLUDING REMARKS . . . . . . . 325
INTRODUCTION
Peroxisomes belong to the microbody fam-
ily, with glyoxysomes and glycosomes as theother members, and represent a class of ubiq-
uitous and essential cell organelles characterized by the presence of a proteinaceous matrix
surrounded by a single membrane. Since this
topic was last reviewed in this journal in 1992(1), our knowledge about the biochemistry o
peroxisomes has increased substantially for anumber of different reasons. First, the identi-
fication and characterization of yeast mutantsdefective in peroxisome biogenesis have al-
lowed the resolution of the principal features
of peroxisome biogenesis, which includes thetargeting of peroxisomal matrix proteins via
one of two distinct peroxisomal targetingsignals (PTS1 and PTS2). This knowledge
has been used to perform computer-basedsearches for proteins that contain a PTS1 or a
PTS2 notably in the yeastSaccharomyces cere-
visiae and to identify the corresponding mam-malian orthologues, using homology probing
This strategy, together with more classical ap-proaches, such as protein purification, has led
to the identification of a series of peroxisomaproteins. Second, the recognition of a large
class of genetic diseases in man, in which ei-
ther peroxisome biogenesis per se or a certainperoxisomal function is defective, has pro-
vided new insights into the metabolic role ofperoxisomes in humans. As a result of these
combined studies, it is nowclear that fatty acid(FA) beta-oxidation is a general feature of vir-
tually all types of peroxisomes. In addition
peroxisomes in higher eukaryotes, includinghumans, catalyze a number of additional per-
oxisomal functions not shared by peroxisomesin lowereukaryotes, including etherphospho
lipid biosynthesis, FA alpha-oxidation, andglyoxylate detoxification.
Another major step forward has been
the discovery that, in contrast to the long-held view that the peroxisomal membrane
is freely permeable to low-molecular-weightsubstances, peroxisomes are in fact closed
structures under in vivo conditions. This
296 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
3/41
requires the presence of peroxisomal mem-
brane proteins, which allow the specific en-trance and exit of metabolites. Some progress
has been made in this respect with the iden-tification of a set of half ATP-binding cas-
sette (ABC) transporters, possibly involved intransmembrane FA transport.
In this review, we present the current stateof knowledge about (a) the biochemistry ofperoxisomes, with special emphasis on the en-
zymology and metabolic functions of mam-mals, notably in humans; (b) the transport
properties of mammalian peroxisomal mem-branes; and (c) the biochemistry in patients
suffering from different peroxisomal disor-
ders and corresponding mouse models.
PEROXISOMAL PROTEINS
It has become clear that peroxisomes are in-volved in a variety of metabolic pathways,
which implies the presence of a large number
of proteins in the peroxisomal matrix. Belowwe define criteria for the peroxisomal localiza-
tion of proteins and discuss some commonlyused strategies to demonstrate the peroxiso-
mal localization for a given protein.
When to Call a Protein aPeroxisomal Protein?
It is estimated that mammalian peroxisomescontain some 50 different enzyme activities,
many of which have been attributed to es-
tablished peroxisomal proteins (Table 1). Inaddition, a number of peroxisomal proteins
have been reported without a known catalyticactivity. This includes the peroxisomal pro-
tein PeP, encoded byFNDC5, with no signif-icant homology to any known protein except
for a short stretch of amino acids containingthe fingerprint of the fibronectin type III su-perfamily (2). In addition, a number of per-
oxisomal proteins have been identified witha catalytic activity of unknown function, as,
for example, the peroxisomal nudix hydrolaseNudt7 (3). Furthermore, some enzyme activ-
ities are not yet linked to a specific peroxiso-
FA: fatty acid
mal protein. These activities may be catalyzed
by new, yet unidentified peroxisomal proteinsor may be a side reaction of a known pro-
tein, as shown, for instance, for 2,4-dienoyl-coenzyme A (CoA) reductase, which also cat-
alyzes the NADPH-dependent reduction ofretinal to retinol (4), at least in vitro, and the
peroxisomal enzyme D-bifunctional protein,which has abundant 17 beta-estradiol dehy-drogenase activity under in vitro, but not in
vivo, conditions. The latter data further im-ply that the identification of a certain enzyme
activity in peroxisomes under in vitro condi-tions does not necessarily imply that peroxi-
somes also catalyze this activity under in vivo
conditions.Most enzyme activities listed in Table 1
are unique to peroxisomes, but some are
shared with other subcellular compartments,including the mitochondria and cytosol. Sucha multiple subcellular localization may be due
to the existence of different isoforms of a pro-
tein targeted to different subcellular sites, as,for example, is the case for the enzymes in-
volved in FA beta-oxidation in mammals (per-oxisomal and mitochondrial), or the presence
of multiple targeting signals within the sameprotein, as, for example, shown for 3-hydroxy-
3-methylglutaryl-CoA lyase (5) and alpha-
methylacyl-CoA racemase (AMACR) (68).These enzymes are located in both peroxi-
somes and mitochondria.Conclusive evidence for the peroxiso-
mal localization of a certain protein and/orits activity requires the actual identification
and characterization of the protein and the
demonstration of its physical presence insidethe organelles. In this respect, it should be
noted that the peroxisomal localization of acertain protein may be species dependent.
Furthermore, the finding of a peroxisomal lo-calization in one species does not necessarily
mean that its homologue is also peroxisomal
in other species. A well-documented exampleof this is alanine:glyoxylate aminotransferase
(AGT), which is peroxisomal in humans, rab-bits, and guinea pigs, has a dual peroxiso-
mal and mitochondrial location in rats and
www.annualreviews.org Biochemistry of Peroxisomes 297
-
7/30/2019 New Per Ox i Some Complete
4/41
Table1
Listofperoxisomalenzymesandotherperoxisomalproteinsfromhumansandtheirperoxisomaltargetingsequences(PT
S1orPTS2)
Peroxisomal(enzyme)protein
Gene
symbol
Enzyme
symbol
EC
number
Humanlocus
Targeting
signal
PTS1
or
PTS
2
Targeting
sequence
Peroxisomalbeta-oxidation
Acyl-CoAoxidase1(palmitoyl-CoAoxidase)
AC
OX1
ACOX1
1.3.3.6
17q25
PTS1
-SKL
Acyl-CoAoxidase2(branched-chainacyl-CoAoxidase)
AC
OX2
ACOX2
1.3.3.6
3p14.3
PTS1
-SKL
Acyl-CoAoxidase3(pristanoyl-CoAoxidase)
AC
OX3
ACOX3
1.3.3.6
4p15.3
PTS1
-SKL
L-bifunctionalprotein(
peroxisomalmultifunctional
enzyme1)
EH
HADH
LBP/MFP1
1.1.1.35;
5.3
.3.8;
4.2
.1.17
3q26.33q28
PTS1
-SKL
D-bifunctionalprotein(peroxisomalmultifunctional
enzyme2)
HSD17B4
DBP/MFP2
4.2.1.-;
1.1
.1.35
5q2
PTS1
-AKL
Peroxisomalbeta-ketoth
iolase1(straight-chainthiolase)
AC
AA1
2.3.1.16
3p23-p22
PTS2
-RLQVVLGHL
Peroxisomalbeta-ketoth
iolase2(branched-chain
thiolase)
SC
P2
SCP2
1p32
PTS1
-AKL
Alpha-methylacyl-CoAracemase
AM
ACR
AMACR
5.1.99.4
5p13.2q11.1
PTS1
-(K)ASL
Carnitineacetyltransferase
CR
AT
CAT
2.3.1.7
9q34.1
PTS1
-AKL
Carnitineoctanoyltransferase
CR
OT
COT
7q21.1
PTS1
-THL
Delta3,5-,delta2,4-dien
oyl-CoAisomerase
EC
HI
19q13.1
PTS1
-SKL
Peroxisomal2,4-dienoyl-CoAreductase2
DECR2
16p13.3
PTS1
-AKL
Peroxisomal3,2-trans-e
noyl-CoAisomerase
PE
C1
6p24.3
PTS1
-SKL
Very-long-chainacyl-CoAsynthetase
SL
C27A2
VLCS
6.2.1.-
15q21.2
PTS1
-LKL
Acyl-CoAthioesterase2
PT
E1
3.1.1.2
20q12q13.1
PTS1
-SKL
Acyl-CoAthioesterase1
B
PT
E2
3.1.1.2
14q24.3
PTS1
-SKV
Peroxisomaltrans-2-eno
yl-CoAreductase(NADPH)
PE
CR
2q35
PTS1
-AKL
Peroxisomalalpha-oxida
tion
Phytanoyl-CoA2-hydro
xylase
PH
YH/PAHX
PHYH/PAHX
1.14
.11.18
10pterp11.2
PTS2
-RLQIVLGHL
2-Hydroxyphytanoyl-CoAlyase
HPCL2
HPCL2
3p25.1
PTS1
-(R)SNM
Plasmalogenbiosynthesis
Dihydroxyacetonephosphateacyltransferase
GNPAT
DHAPAT
2.3.1.42
1q42.1142.3
PTS1
-AKL
Alkyldihydroxyacetonephosphatesynthase
AG
PS
ADHAPS
2.5.1.26
2q31
PTS2
-RLRVLSGHL
Fattyacyl-CoAreductase1
MLSTD2
FAR1
11p15.2
Fattyacyl-CoAreductase2
MLSTD1
FAR2
12p11.22
298 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
5/41
Glyoxylatemetabolism
Alanine:glyoxylateamin
otransferase
AG
XT
AGT
2.6.1.44;
2.6
.1.55
2q36q37
PTS1
-KKL
Lysinemetabolism
Peroxisomalsarcosineo
xidase/L-pipecolateoxidase
PIPOX
PIPOX
17q11.2
PTS1
-AHL
Oxygenmetabolism
Catalase
CA
T
CAT
1.11
.1.6
11p13
PTS1
-(K)ANL
PeroxiredoxinV(PMP2
0)
PR
DX5
PROX5/PMP20
1.11
.1.7
11q13
PTS1
-SQL
d-aminoacidoxidase
DAO
DAOX
1.4.3.3
12q24
PTS1
-SHL
d-aspartateoxidase
DDO
DASPOX
1.4.3.1
6q21
PTS1
-(K)SNL
Glycolateoxidase(hydroxyacidoxidase1)
HAO1
GOX/HAO1
1.1.3.15
20p12
PTS1
-SKI
Hydroxyacidoxidase2
HAO2
HAO2
1.1.3.15
1p13.3p13.1
PTS1
-SRL
Hydroxyacidoxidase3
HAO3
HAO3
1.1.3.15
-
PTS1
-SRL
Epoxidehydrolase
EP
HX2
EPH2
3.3.2.3
8p21-p12
PTS1
-SKM
GlutathioneS-transferaseclassKappa
GSTK1
GSTK1
7
PTS1
-ARL
Polyaminemetabolism
N1-acetylspermine/sper
midineoxidase
PA
OX
PAO
10q26.3
PTS1
-(R)PRL
Additional(enzyme)pro
teins
Malonyl-CoAdecarboxylase
MLYCD
4.1.1.9
16q12
PTS1
-SKL
3-Hydroxy-3-methylglu
taryl-CoAlyase
HMGCL
HL
4.1.3.4
1p36.1p35
PTS1(+
MTS)
-CKL
Isocitratedehydrogenase(NADP+-linked)
ID
H1
IDH1
1.1.1.42
2q33.3
PTS1
-AKL
NudixhydrolasespecificforCoA
NUDT7
NUDT7
16q23.1
PTS1
-SRL
Insulin-degradingenzym
e
ID
E
IDE
3.4.24.56
10q23q25
PTS1
-AKL
Serinehydrolaselike
SE
RHL
22q13.2
q13.31
Lonprotease
LO
NP
16q12.1
PTS1
-SKL
Nudix-typemotif19(Roswell-Parkcomplex2)
D7RP2e
RP2p
19q13.11
PTS1
-SHL
Trim37
TR
IM37
6p21.3
PeP
FN
DC5
FNDC5/PeP
1p35.1
PTS1
-SKI
PMP22
PX
MP2
PMP22
12q24.33
www.annualreviews.org Biochemistry of Peroxisomes 299
-
7/30/2019 New Per Ox i Some Complete
6/41
marmosets, and is mitochondrial in cats (9).
Another example is the localization of the FAbeta-oxidation pathway, which is exclusively
peroxisomal in yeast but shows a dual peroxi-somaland mitochondrial localization in mam-
mals and plants (10).
Strategies to Identify PutativePeroxisomal Proteins
Both forward and reverse genetics approacheshave been employed to identify putative per-
oxisomal proteins. In short, the forward ge-netics approach involves the conventional
purification of a protein, which exhibits a cer-
tain enzyme activity assumed to be perox-isomal, followed by the generation of spe-
cific antibodies against the purified protein,
which can be used to demonstrate its actualsubcellular localization. Furthermore, the en-coding cDNA and corresponding gene can
be identified by a degenerated polymerase
chain reaction approach that is based on apartial amino acid sequence of the purified
protein. The reverse genetics approach be-came popular because of the availability of the
genomic sequences of various organisms, in-cluding yeasts, mice, and humans, in conjunc-
tion with the increased knowledge of func-
tional domains within amino acid sequences(e.g., catalytic sites and targeting signals), al-
lowing selective database searches for genesencoding putative peroxisomal proteins with
certain activities. This in silico strategy hasbeen very successful in identifying novel puta-
tive peroxisomal proteins [e.g., proteins with
consensus peroxisomal targeting signals (11)]as well as orthologues (i.e., functional homo-
logues identified on the basis of significant se-quence similarity) of proteins determined to
be peroxisomal in other species, a strategy alsoreferred to as homology probing (12, 13).
Strategies to Demonstrate thePeroxisomal Localization of Proteins
The most important criterion for the per-
oxisomal localization of a certain protein
remains that the protein should be shownas physically present inside peroxisomes
This is true particularly for candidate per-
oxisomal proteins identified by the reversegenetics approach, because the presence of a
consensus PTS in the amino acid sequencei.e, a PTS1-consensus sequence: (S/A/C)
(K/R/H)-(L/M) or a PTS2-consensus sequence: (R/K)-(L/I/V)-X5-(Q/H)-(L/I/V)does not necessarily mean that the protein
is truly peroxisomal. For example, althoughboth pristanoyl-CoA oxidase and the bile acid
conjugating (BACAT) enzyme harbor thesame C-terminal PTS1-like SQL tripeptide
only pristanoyl-CoA oxidase is peroxisoma
and BACAT cytosolic (14, 15). Also, despitethe presence of a consensus PTS1 (-SRL)
phosphomevalonate kinase was recently
demonstrated to be a cytosolic protein (16).Several approaches can be employed
to demonstrate a peroxisomal localization
These include conventional subcellular frac-
tionation studies by which tissues or cells arefirst homogenized and separated by differ-
ential centrifugation into organelle-enrichedfractions that are further fractionated by den-
sity gradient centrifugation. The fractions ofthese gradients are then analyzed by specific
enzyme activity measurements and/or im-
munoblotanalysis,using specific antibodies todeterminethe profileof the protein of interes
in the gradient in comparison to the profileof established marker proteins/enzymes for
the various subcellular organelles. As activitymeasurements may not always be specific be-
cause enzymes often can handle multiple sub-
strates, the combined approach with specificantibodies is highly recommended.
The peroxisomal localization of solubleproteins can also be studied through con-
trolled permeabilization of cellular mem-branes with digitonin. Because digitonin per-meabilizes cellular membranes forming a
complex with cholesterol and organelle mem-branes contain lower levels of cholestero
than the plasma membrane, this treatmentleads to differential leakage of proteins. This
leakage can be assessed by determining the
300 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
7/41
releaseoftheproteinofinterestincomparison
to that of established marker proteins usingspecific enzyme activity measurements and/or
immunoblot analyses using specific antisera(see, for examples, References 16, 17, and 18).
Conclusive evidence for the peroxisomallocalization of a protein can be obtained by
in situ (immuno) microscopical techniques,including immunoelectron microscopy, im-munofluorescence microscopy, and immuno-
histochemistry. Because the outcome of thesetechniques relies heavily on the quality of an-
tisera used to detect the protein, it is essen-tial to obtain antisera that are highly spe-
cific and exclusively recognize the protein
of interest under native conditions. The im-portance of performing immunolocalization
studies to establish the peroxisomal localiza-
tion of a certain protein using specific anti-bodies has been shown on numerous occa-sions. This is exemplified by recent studies
by Yokota and coworkers (19), showing that
the NADP-linked isocitrate dehydrogenaseencoded by IDH1 is predominantly, if not
exclusively, peroxisomal, whereas subcellu-lar fractionation studies, based on differen-
tial centrifugation of homogenates, revealedthat the enzyme is predominantly cytosolic
and only partially peroxisomal (19).
The functionality of putative PTS1 orPTS2 motifs in the amino acid sequence of
a protein can be tested by reporter studiesin which the protein or portions thereof are
fused to a reporter protein such as green fluo-rescent protein or specific epitopes (e.g., myc,
HA). This allows easy detection of the protein
constructs upon expression in cells. It shouldbe noted, however, that the observation that a
certain (truncated) amino acid sequence is ca-pable of targeting a reporter to peroxisomes
does not necessarily imply that it also func-tions as a true PTS in the authentic protein if
the latter has not been shown to actually re-
side in peroxisomes by other means. Indeed,a putative C-terminal PTS1 may not be func-
tional when, for example, the PTS1 is hiddenwithin the three-dimensional structure of the
protein or when, in addition, an N-terminal
mitochondrial targeting sequence is presentin the same protein. Furthermore, great care
should be taken with the interpretation of re-
sults obtained with reporter studies becausein most cases these involve overexpression,
which may introduce unpredictable artifacts(16, 20). It should be noted that the specific
context of a putative PTS is important fortargeting, by increasing the affinity between
the PTS-containing peptide and its receptor
(2124). For instance, it has been shown forcatalase, which ends in ANL, a weak PTS1,
that the lysine present in the (4) position ofcatalase (-KANL) greatly stimulates binding
to the PTS1-receptor (22). Optimized aminoacid residues at positions (4) and (5) can en-
hance affinities by at least two orders of mag-
nitude (23).
SELECTED METABOLICPATHWAYS
Below we describe the role of peroxisomes inselected metabolic pathways.
Oxygen Metabolism, ReactiveOxygen Species, and Reactive
Nitrogen Species Metabolism
Peroxisomes harbor a number of oxidases thatreduce O2 to H2O2 (25). The H2O2 produced
can be disposed of via several enzymes, in-cluding catalase, glutathione peroxidase, and
peroxiredoxin V (PMP20). The decomposi-
tion of H2O2 by catalase may occur catalyt-ically (2H2O2 O2 + 2H2O) or peroxi-
datically (H2O2 + AH2 A+ 2H2O), inwhich the conversion of one molecule H2O2to two molecules of H2O is coupled to the ox-idation of different hydrogen donors (AH2),
such as ethanol, methanol, formaldehyde, for-mate, and nitrite. In addition to catalase, per-oxisomes also contain glutathione peroxidase
activity (26). A third peroxisomal enzyme thatremoves H2O2 is PMP20 (27), which exhibits
thiol-specific antioxidant activity.Apart from H2O2, peroxisomal en-
zymes also generate other reactive species,
www.annualreviews.org Biochemistry of Peroxisomes 301
-
7/30/2019 New Per Ox i Some Complete
8/41
including superoxide anions. One source of
superoxide anions is xanthine oxidoreductase,which can exist in two forms, including
a dehydrogenase and oxidase form (thelatter form generates superoxide anions).
Angermuller et al. (28) were the first toidentify xanthine oxidase activity in the core,
but not in the matrix, of peroxisomes. Recentstudies in rat liver in which use was made ofimproved methods applied to unfixed cryostat
sections have shown that xanthine oxidase isnot only present in the core of peroxisomes
but also in the peroxisomal matrix (29).Furthermore, xanthine oxidase appears to be
the predominant, if not exclusive, form of
xanthine oxidase in peroxisomes, whereas inthe cytosol, the reverse is true with xanthine
dehydrogenase predominating over xanthine
oxidase.Inactivation of superoxide anions is
brought about by superoxide dismutases. Sev-
eral reports have shown the presence of
Cu/Zn-SOD (30, 31) and Mn-SOD activi-ties (32) in peroxisomes, although theproteins
responsible for these activities remain to beidentified. It has also been claimed recently
that peroxisomes contain inducible nitric ox-ide (NO) synthase activity (33), which, if true,
would be an important intraperoxisomal gen-
erator of NO species. Together with super-oxide anions, NO would generate peroxyni-
trite, a highly reactive species. Interestingly,peroxiredoxin V, which has been localized to
peroxisomes (see Table 1) as well as to mito-chondria and cytosol, was recently shown to
exhibit potent peroxynitrite reductase activity
(34).Peroxisomes also contain epoxide hydro-
lase activity (3537). Epoxides are a group ofhighly reactive molecules of both exogenous
and endogenous origin. Some of the most po-tent carcinogenic and mutagenic compounds
only become active when transformed into
their epoxides. Because they are very elec-trophilic, they easily react with nucleophilic
groups such as lipids containing unsaturatedFAs, DNA, RNA, and proteins. Epoxides,
which can be synthesized endogenously, in-
clude epoxides of prostaglandins, leukotriensarachidonic acid, cholesterol, and unsaturated
FAs.Onesinglegene coding fora protein with
a weak PTS1 signal (SKI) has been identifiedwhich gives rise to a bicompartmental distri-
butionof epoxide hydrolasein both theperox-isomes and cytosol (36). According to others
however, the peroxisomal epoxide hydrolaseis different from that present in other com-
partments (37).
Finally, peroxisomes also contain glu-tathione S-transferase (GST) activity (38)
GSTs catalyzethe conjugation of electrophilicsubstrates to glutathione but, in addition, have
reduced glutathione-dependent peroxidaseand isomerase activities. The GST identified
in peroxisomes belongs to the kappa family
The true function of this GSTK1 remains to
be identified, however. The enzyme shows reactivity with 1-chloro-2,4-dinitrobenzene as
well as with cumene hydroperoxide and 15-S-
hydroperoxy-5,8,11,13-eicosatetraenoic acid(38).
Ether-Phospholipid Biosynthesis
Ether phospholipids may occur in two
forms, including (a) plasmanyl-phospholipidand (b) plasmenyl-phospholipids (plasmalo-
gens) with a 1-O-alkyl and 1-O-alk-11-enyether bond, respectively, and usually con
tain ethanolamine or choline as head groupwith ethanolamine predominating in humans
(fourfold). In humans, plasmalogens make up
some 18% of total phospholipid mass andshow a cell- and tissue-specific distribution
High levels of plasmenyl ethanolamine occurin brain, heart, lung, kidney, spleen, skele-
tal muscle, and testis, whereas high levels oplasmenyl choline occur in heart and skele-
tal muscle with very low levels in all othertissues. Macrophages and neutrophils contain not only high plasmenyl ethanolamine
levels, but also significant levels of the satu-rated ether-phospholipid plasmanyl choline
which is used by these cells for the produc-tion of platelet-activating factor (1-O-alkyl-2
acyl-sn-glycero-3-phosphocholine). The sn-1
302 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
9/41
position of plasmalogens is occupied predom-
inantly by C16:0, C18:0, and C18:1 fatty al-cohols, whereas the sn-2 position of ether
phospholipids usually contains polyunsatu-rated FAs.
Peroxisomes and the enzymology of ether-
phospholipid biosynthesis. The first com-mitted step in the biosynthesis of ether-linkedglycerolipids is the formation of the ether
linkage by the enzyme alkyldihydroxyace-tone phosphate synthase (alkyl-DHAP syn-
thase or ADHAPS) (Figure 1a) encoded bythe AGPS gene. In this reaction, the ester-
linked FA of acyl-DHAP is replaced by a fatty
alcohol with an ether bond, forming alkyl-DHAP. The reaction proceeds by a ping-pong
mechanism: acyl-DHAP first binds to the en-
zyme, followed by release of the FA, result-ing in an activated enzyme-DHAP complex,
which then reacts with a fatty alcohol to pro-
duce alkyl-DHAP. Alkyl-DHAP synthase is
an established peroxisomal enzyme (3941),which can react with a range of fatty alco-
hols, including saturated (C10:0 to C18:0) aswell as mono- (C18:1) and polyunsaturated
(C18:2 and C18:3) alcohols, and this contrastsmarkedly to the fatty alcohols found in plas-
malogens, which are C16:0, C18:0, and C18:1
only (see below). Alkyl-DHAP synthases havebeen identified in various eukaryotic species
and represent one of the few peroxisomal en-zymes equipped with a PTS2-targeting se-
quence in all organisms exceptCaenorhabditis
elegans. In this organism, the enzyme contains
a PTS1, which is in line with the notion that
thePTS2 pathway is missing in C. elegans(42).The two substrates of alkyl-DHAP syn-
thase, i.e., acyl-DHAP and a long-chainfatty alcohol, are also generated by peroxi-
somes (Figure 1b). Acyl-DHAP is synthe-sized from DHAP and an acyl-CoA esterby the peroxisomal enzyme dihydroxyace-
tone phosphate acyltransferase (DHAPAT)encoded by GNPAT. The enzyme can han-
dle only a small range of acyl-CoAs, includingsaturated (C14:0 and C16:0) and unsaturated
(C18:1) acyl-CoAs (43), shows a broad pH
Figure 1
(a) Schematic representation of the steps involved in the biosynthesis of (phosphatidylcholine) and PE (phosphatidylethanolamine) plasmalogensand (b) the topology of the enzymes involved in the biosynthesis of
plasmalogens. Abbreviations used: AADHAPR, acylalkyl-dihydroxyacetophosphate reductase; ADHAPS, alkyl-DHAP synthase; DHAPAT,dihydroxyacetone phosphate acyltransferase; FAR, fatty acyl-CoAreductase; G3PDH, glycerol-3-phosphate dehydrogenase; and VLCS,
very-long-chain acyl-CoA synthetase.
CHO: Chinesehamster ovary
optimum between 7.0 and 9.0, and is mem-
brane associated, with its catalyticsite exposedto the peroxisome interior. All DHAPAT
amino acid sequences known from differenteukaryotic species contain a PTS1 sequence
(44, 45). DHAPAT is crucial for plasmalo-gen synthesis because DHAPAT-deficient hu-man and Chinese hamster ovary (CHO) cell
lines are unable to synthesize plasmalogens.Interestingly, acyl-DHAP can also be syn-
thesized outside peroxisomes by other acyl-transferases including microsomal G3PAT
(46), but this acyl-DHAP is not available for
www.annualreviews.org Biochemistry of Peroxisomes 303
-
7/30/2019 New Per Ox i Some Complete
10/41
Figure 1
(Continued)
peroxisomal alkyl-DHAP synthase, mostlikely because acyl-DHAP synthesized out-
side peroxisomes is unable to traverse the
peroxisomal membrane (Figure 1b). Alkyl-DHAP synthase and DHAPAT form a 210-
kDa heterotrimeric complex within peroxi-
somes (44, 47) (Figure 1b). DHAPAT is onlystable inside peroxisomes and when present inthe 210-kDa complex, whereas alkyl-DHAP
synthase is stable in peroxisomes and active
even in the absence of DHAPAT (48).Although some of the long-chain fatty
alcohols required for the alkyl-DHAP syn-thase reaction maycome from dietary sources,
the bulk is synthesized from acyl-CoAs. Thetwo consecutive reductions required to trans-
form acyl-CoAs into the corresponding al-
cohols (acyl-CoA aldehyde alcoholare catalyzed by the same fatty acyl-CoA re-
ductase (FAR), which does not release the
intermediate aldehyde and has NADPH asthe preferred cosubstrate. In developing ratbrain, a long-chain acyl-CoA reductase ac-
tivity was identified as reactive with C16:0-
C18:0-, and C18:1-CoA only (49). The en-zyme was specifically localized in the perox-
isomal membrane with its catalytic site ex-posed to the cytosol (49) (see Figure 1b)
304 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
11/41
On the basis of a specific substrate spec-
trum, it was speculated that this enzyme is re-sponsible for the virtually exclusive presence
of C16:0, C18:0, and C18:1-alk(en)yl chainsat the sn-1 position of ether phospholipids.
Recently, the identification of two acyl-CoAreductases, called FAR1 and FAR2, with dif-
ferent substrate specificities and tissue distri-butions was reported (50). Both FAR1 andFAR2 are peroxisomal membrane proteins,
but they lack clear transmembrane-spanningregions as well as PTS1 or PTS2 sequences.
FAR1 is reactive with saturated and unsatu-rated acyl-CoAs of 1618 carbons, whereas
FAR2 prefers saturated acyl-CoAs of 16 or 18
carbon atomsonly. FAR1 expression was iden-tified in many mouse tissues, with the highest
level in the preputial gland, a modified seba-
ceous gland. FAR2 expression was more re-stricted in distribution and most abundant inthe eyelid, which contains wax-laden meibo-
mian glands. Both FAR1andFAR2expression
was observed in the brain, a tissue rich in etherlipids. These findings suggest that fatty alco-
hol synthesis in mammals is accomplished bytwo FAR enzymes (FAR1and FAR2), encoded
byMLSTD2 and MLSTD1, respectively, andexpressed at high levels in tissues known to
synthesize wax esters and ether lipids (50).
The last contribution of peroxisomes toether-phospholipid biosynthesis is the re-
duction of alkyl-DHAP, generated by alkyl-DHAP synthase, to alkyl-G3P. The respon-
sible enzyme acyl/alkyl-DHAP reductase ismembrane bound, faces the cytosol both in
peroxisomes and in the endoplasmatic reticu-
lum, and preferentially reacts with NADPHrather than NADH (51). All subsequent
steps occur in the endoplasmic reticulum(Figure 1b) (see References 46 and 52 for re-
cent reviews).The physiological role of ether phos-
pholipids, including plasmalogens, has not
been established with certainty. They havebeen implicated in membrane dynamics, in-
tracellular signaling, cholesterol transport andmetabolism, oxidative stress, and polyunsat-
urated FA metabolism (46, 5358). The fact
that isolated deficiencies of DHAPAT andalkyl-DHAP synthase in humans are associ-
ated with severe clinical abnormalities and
early death (see the section on peroxisomaldisorders) indicates that ether phospholipids
are essential for life.
Peroxisomal Fatty AcidBeta-Oxidation
In contrast to most other functions of per-oxisomes, which may vary between different
species and within specific cell types in a sin-gle species, FA beta-oxidation is a universal
property of peroxisomes in most, if not all, or-
ganisms. In yeast and plants, peroxisomes arethe sole site of FA beta-oxidation, whereas in
higher eukaryotes beta-oxidation may occur
in both mitochondria and peroxisomes, fol-lowing a mechanism involving dehydrogena-tion, hydration, dehydrogenation again, and
thiolytic cleavage as depicted in the panels of
Figure 2a,b for mitochondrial beta-oxidationand peroxisomal beta-oxidation, respectively.
Although similar in mechanism, mitochon-drial and peroxisomal beta-oxidation fulfill
different functions, as concluded from theusually severe but different clinical signs and
symptoms associated with inherited defects
in either mitochondrial (59) or peroxisomalbeta-oxidation (60).
FAs destined for beta-oxidation may orig-inate from outside the cell or result from
intracellular breakdown of lipids, for in-stance in lysosomes. Extracellular FAs prob-
ably enter cells by a saturable mechanism
mediated by candidate proteins, such as theplasma membrane FABPPM, the FA translo-
case (FAT/CD36), as well as one or moremembers of the FA transport protein (FATP)
family of molecules, which have been hypoth-esized to harbor both FA transport as wellas acyl-CoA synthetase activity (6163). FAs
generated within the cell may be activatedby one of the acyl-CoA synthetase enzymes
(Acs1-6), which activate different FAs withunique efficiencies (64). Once activated, FAs
cannot repartition back into the membrane
www.annualreviews.org Biochemistry of Peroxisomes 305
-
7/30/2019 New Per Ox i Some Complete
12/41
Figure 2
Schematic representation of the mitochondrial and peroxisomalbeta-oxidation systems in humans. (a) In mitochondria, the FADH2 andNADH, generated in the first and third steps of beta-oxidation, aredirectly reoxidized by the respiratory chain (RC), (b) whereas inperoxisomes, molecular oxygen is the electron acceptor in the first step ofbeta-oxidation, resulting in the formation of H2O2, which is reconvertedinto O2 by catalase. The NADH generated in the third step ofperoxisomal beta-oxidation is reoxidized via a NAD(H)-redox shuttle,
involving the cytosolic and peroxisomal isoforms of malate dehydrogenasein S. cerevisiae and lactate dehydrogenase in higher eukaryotes.
VLCFA:very-long-chain fattyacid
because of their decreased hydrophobicity.The activation also ensures low unesterified
FA levels in the cell, thereby maintaining aconcentration gradient that is favorable for
the entry of more unesterified FAs into the
cytosol. The major differences between per-oxisomal and mitochondrial beta-oxidation
include different substrate specificities and
transport of substrates and products of beta-oxidation across the membrane (see Refer-
ences 65 and 66 for reviews).
Different substrate specificities. Short-and medium-chain FAs are exclusively andlong-chain FAs are predominantly beta-
oxidized in mitochondria, whereas very-long-chain FAs (VLCFAs), notably 26:0
can only be handled by peroxisomesOther substrates handled only by per-
oxisomes are (a) pristanic acid (2,4,6,10-
tetramethylpentadecanoic acid), derived fromdietary sources such as pristanic acid itself
or its precursor phytanic acid, which is con-
verted to pristanic acid by alpha-oxidation; (b)di- and trihydroxycholestanoic acid (DHCAand THCA), produced from cholesterol in
the liver and converted to chenodeoxycholic
and cholic acid, respectively, after one cy-cle of beta-oxidation in the peroxisome
(c) long-chain dicarboxylic acids, producedby omega-oxidation of long-chain monocar-
boxylic acids; (d) certain polyunsaturated FAsincluding tetracosahexaenoic acid (C24:6)
which undergoes one cycle of beta-oxidation
in peroxisomes to produce docosahexaenoicacid (C22:6); (e) certain prostaglandins and
leukotrienes; ( f) some xenobiotics; and (g)vitamins E and K.
Transport of substrates and products of
beta-oxidation across the membrane. In
the case of mitochondria, long-chain FAs(LCFAs) enterthe mitochondrialspacevia the
carnitine cycle (Figure 2a), whereas short-and medium-chain FAs enter directly in their
protonated form. For peroxisomes, the situa-tion is less clear, but a carnitine-mediated im-port mechanism has been ruled out (66). As
discussed below, the FAs destined for beta-oxidation in peroxisomes probably enter per-
oxisomes as acyl-CoA esters. Oxidation oFAs in peroxisomes generates a number of
acyl-CoA esters, including (a) medium-chain
306 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
13/41
Figure 2
(Continued)
acyl-CoAs, e.g., 4,8-dimethylnonanoyl-CoAin the case of pristanoyl-CoA beta-oxidation;
(b) proprionyl-CoA, and (c) acetyl-CoA. The
fate of each of these products may vary amongdifferent organs and cell types. In principle,
LCFA: long-chaifatty acid
there are different ways in which these CoAesters canbe metabolized further (Figure 2b).
First, different acyl-CoAs can be con-
verted into the corresponding carnitine es-ters via the peroxisomal enzymes carnitine
www.annualreviews.org Biochemistry of Peroxisomes 307
-
7/30/2019 New Per Ox i Some Complete
14/41
acetyltransferase and carnitine octanoyltrans-
ferase, as encoded by CRAT and CROT, re-spectively, followed by export from the per-
oxisomes and uptake into the mitochondrionvia the carnitine acylcarnitine carrier as was
shown for acetyl-CoA and propionyl-CoA(67) and 4,8-dimethylnonanoyl-CoA (68) in
cultured skin fibroblasts. Furthermore, acyl-CoA esters may be hydrolyzed within theperoxisome by one of the peroxisomal acyl-
CoA thioesterases (69), yielding the free acidand CoA (Figure 2b). In hepatocytes, the
thioesterase route is very active, with acetateas a major product of the acetyl-CoA units
produced in peroxisomes (70). Recent studies
on the peroxisomal and mitochondrial oxida-tion of FAs have shown that there is no de-
tectable transfer of peroxisomal acetyl-CoA
units to the mitochondrion for oxidation toCO2 and H2O, at least in perfused rat hearts(71). This implies that, in contrast to the
liver, peroxisomal FA oxidation in the heart is
not accompanied by the hydrolysis of acetyl-CoA and release of acetate. The exact fate of
the acetyl-CoA units produced in heart per-oxisomes remains to be determined. Recent
studies in HepG2 cells have shown that theacetyl-CoA units generated in liver peroxi-
somes are not only converted into acetate (70)
but are also used for chain elongation (72)(see Figure 2b).
Enzymology of the peroxisomal beta-
oxidation system. Saturated unbranched
and 2-methyl-branched FAs are the onlyFAs that can undergo direct beta-oxidation.
In contrast, other FAs, such as mono- andpolyunsaturated FAs, 3-methyl branched-
chain FAs, and 2-hydroxy FAs, first needto undergo remodeling before they become
substrate for peroxisomal beta-oxidation(Figure 3). The first step of beta-oxidation inmammalian peroxisomes is catalyzed by dif-
ferent acyl-CoA oxidases, with important dif-ferences between the rat and human. Extra-
hepatic peroxisomes in the rat contain twoacyl-CoA oxidases, including palmitoyl-CoA
oxidase (ACOX1) and pristanoyl-CoA oxi-
dase (ACOX3), whereas liver peroxisomescontain an additional cholestanoyl-CoA ox-
idase (ACOX2), specifically reacting with the
CoA esters of the bile acid intermediatesDHCA and THCA (73). Rat ACOX1 is ac-
tive with CoA esters of straight-chain mono-and dicarboxylic FAs, prostaglandins, VLC
FAs, and xenobiotics, whereas rat ACOX3is active with 2-methyl-branched-chain acyl-
CoAs, such as pristanoyl-CoA, but also
handles long and very-long straight-chainacyl-CoAs (73). In the rat, only ACOX1 is in-
ducible by peroxisome proliferators. Interest-ingly, human peroxisomes contain only two
oxidases; the first one is palmitoyl-CoA ox-idase, the counterpart of rat ACOX1, with
similar substrate spectrum and molecular
characteristics (74). The second human per-
oxisomal oxidase is the branched-chain acyl-CoA oxidase, active with 2-methyl-branchedcompounds, such as pristanoyl-CoA and the
CoA esters of DHCA and THCA, as well
as straight-chain acyl-CoAs, including theCoA esters of VLCFAs and dicarboxylic
acids (74). Cloning of the cDNA for hu-man branched-chain acyl-CoA oxidase re-
vealed that it is the homologue of the raliver-specific cholestanoyl-CoA oxidase (75)
Incontrasttotheratenzyme,however,human
branched-chain acyl-CoA oxidase is ubiqui-tously present in all tissues. Remarkably, the
gene coding for the homologue of rat ACOX3was also identified in the human genome, bu
both immunoblot and northern blot analysesfailed to identify its expression in human tis-
sues (76). It was therefore speculated that the
gene was only expressed under certain, forinstance developmental, conditions. Interest-
ingly, abundant expression of this pristanoyl-CoA oxidase was found recently in human
prostate tissue as well as in some prostate can-cer cell lines (77).
Human, rat, and mouse peroxisomes con-
tain two distinct bifunctional proteins thatdisplay both enoyl-CoA hydratase and 3-
hydroxy-acyl-CoA dehydrogenase activitiesand catalyze the conversion of 2-trans-enoyl-
CoAs to 3-ketoacyl-CoAs. l-bifunctiona
308 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
15/41
Figure 3
Schematic description of the enzymes required for the conversion of different FAs into thecorresponding acyl-CoA esters. Abbreviations used: ALDH, aldehyde dehydrogenase; HPCL2,2-hydroxyphytanoyl-CoA lyase.
protein (LBP) forms and dehydrogenates l-
3-hydroxyacyl-CoAs, andd-bifunctional pro-tein (DBP) forms and dehydrogenates d-
3-hydroxyacyl-CoAs. Alternative names forLBP and DBP are multifunctional enzymes
I and II (perMFE-I and -II) (78), multifunc-tional proteins 1 and 2 (MFP-1 and -2) (79),
and l- and d-peroxisomal bifunctional en-
zyme(l-PBE andd-PBE)(80).Itiswellestab-lished now that DBP is the main, if not exclu-
sive, enzyme involved in the beta-oxidation ofVLCFAs, pristanic acid, as well as DHCA and
THCA (Figure 4). Substrate specificity stud-ies have shown that both LBP and DBP re-
act with straight-chain enoyl-CoAs, whereasonly DBP reacts with the enoyl-CoA estersof pristanic acid, DHCA, and THCA (78, 79,
8184). The importance of DBP in the beta-oxidation of all these compounds has become
clear through the identification and charac-terization of patients with a deficiency of DBP
(85) and by the generation of a DBP (MFP-2)
knockout mouse (86). The physiological roleof LBP remains unclear, although recent stud-
ies indicated that it may be the primary en-
zyme involved in dicarboxylic acid oxidation(87).
Both bifunctional proteins show very littlesequence homology and are structurally very
different. The N-terminal part of LBP con-
tains theenoyl-CoAhydratase activity andtheC-terminal part, the 3-hydroxyacyl-CoA de-
hydrogenase activity. In addition, LBP alsoharbors 3,2-enoyl-CoA isomerase activ-
ity. In contrast, the N-terminal domain ofDBP is responsible for the 3-hydroxyacyl-
CoA dehydrogenase activity, the central partcontains the enoyl-CoA hydratase activity,and the C-terminal domain, sterol carrier pro-
tein (SCP) 2 activity (88).Mammalian peroxisomes also contain
multiple peroxisomal thiolases. Mouse andrat liver peroxisomes contain three dif-
ferent 3-oxoacyl-CoA thiolases, including
www.annualreviews.org Biochemistry of Peroxisomes 309
-
7/30/2019 New Per Ox i Some Complete
16/41
Figure 4
Overview depicting the involvement of the different peroxisomal beta-oxidation enzymes in theperoxisomal beta-oxidation of VLCFAs, pristanic acid (PRIS), DHCA, THCA, tetracosahexaenoic acid(C24:6), and long-chain dicarboxylic acids (DCA). Abbreviation: CoASH, free unesterified coenzyme A.
(a) 3-oxoacyl-CoA thiolase A, (b) 3-oxoacyl-
CoA thiolase B, and (c) SCP-2/3-oxoacyl-CoA thiolase (SCPx). The constitutively ex-
pressed thiolase A and inducible thiolase
B have a virtually identical substrate spec-trum, which is active toward short-, medium-,
long-, and very-long-chain 3-oxoacyl-CoAs,
and are involved in the peroxisomal beta-oxidation of straight-chain FAs (89). SCPxis active toward medium-, long-, and very-
long-chain 3-oxoacyl-CoAs, and it is also re-
active toward the 3-oxoacyl-CoA species of2-methyl branched-chain FAs, such as pris-
tanic acid and the bile acid intermediatesDHCA and THCA (9093). Human per-
oxisomes only contain two thiolases; theseinclude a straight-chain 3-oxoacyl-CoA thi-
olase, encoded by ACAA1, and the SCPx,
encoded by SCP2, which is essential forthe oxidation of 2-methyl branched-chain
FAs, i.e., pristanic acid, DHCA, and THCA(94). The involvement of the different beta-
oxidation enzymes in the oxidation of VL-CFAs, pristanic acid, DHCA, THCA, C24:6,
and long-chain dicarboxylic acids is shown in
Figure 4.
The enzymes described above are neces-
sary and sufficient for the beta-oxidation ofstraight-chain saturated FAs as well as alpha-
methyl branched-chain FAs with the methygroup in the (2S)-configuration. However
auxiliary enzymes are needed for the beta-
oxidation of (2R)-methyl branched-chain FAs
and unsaturated FAs (Figure 3). Oxidationof (2R)-methyl branched-chain FAs requiresthe active participation of the peroxisoma
enzyme 2-methylacyl-CoA racemase, whichis capable of converting (2R)- into (2S)-
branched-chain acyl-CoAs (9597). Interest-
ingly, a single gene (AMACR) codes for a pro-tein equipped with both a mitochondrial and
peroxisomal targeting signal thus explainingits bicompartmental presence in both peroxi-
somes and mitochondria (68). Both the per-
oxisomal and mitochondrial AMACRs are re-quired for the oxidation of pristanic acid (7).
Peroxisomes also contain the full enzy-matic machinery to remove the double bonds
in mono- and polyunsaturated FAs. FAs witha double bond at an even-numbered posi-
tion require the subsequent action of two aux-
iliary enzymes, including 2,4-dienoyl-CoA
310 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
17/41
reductase and 3,2-enoyl-CoA isomerase,to produce the corresponding saturated acyl-
CoA esters. The 2,4-dienoyl-CoA reductase
and 3,2-enoyl-CoA isomerase in perox-isomes are different from their mitochon-
drial counterparts and the products of dis-tinct genes, i.e., the DECR2 (98) and PEC1
(99) genes (see Table 1). Oxidation of FAswith a double bond at an odd-numbered po-sition may proceed via two different path-
ways. Pathway one only involves the 3,2-enoyl-CoA isomerase, whereas pathway two
requires the active participation of three aux-iliary enzymes, including a distinct3,5,2,4-
dienoyl-CoA isomerase, 2,4-dienoyl-CoA
reductase, and 3,2-enoyl-CoA isomerase.After its prior identification in mitochondria,
He et al. (100) detected 3,5,2,4-dienoyl-
CoA isomerase activity in peroxisomes. Sub-sequent studies have shown that a singlegene ECH1 codes for a protein with both
a mitochondrial and peroxisomal targeting
signal, thus explaining the bicompartmen-tal distribution of3,5,2,4-dienoyl-CoA iso-
merase in both mitochondriaandperoxisomes(101).
Peroxisomal Fatty Acid
Alpha-OxidationFAs with a methyl-group at the carbon 3
position are not a substrate for beta-oxidationbut must first undergo alpha-oxidative de-
carboxylation to produce the corresponding
(n-1) FA, with the methyl-group at the 2position, which then can undergo beta-
oxidation. In the past decade, it has becomeclear that, in contrast to FA beta-oxidation,
FA alpha-oxidation is confined to perox-isomes and only accepts acyl-CoA esters
as substrate (Figure 5). Most studies onalpha-oxidation have been performed withthe physiological substrate phytanic acid
(3,7,11,15-tetramethylhexadecanoic acid),which is known to accumulate in different
genetic disorders including Refsum disease(see below). Activation of phytanic acid
to its CoA-ester can occur either outside
the peroxisome by the enzyme long-chainacyl-CoA synthetase (102), present at the
cytosolic face of the peroxisome (103), or
inside the peroxisome by the enzyme very-long-chain acyl-CoA synthetase, a peripheral
peroxisomal membrane protein equippedwith a PTS1-like signal (Figure 5) (104).
Subsequently, phytanoyl-CoA is convertedinto 2-hydroxyphytanoyl-CoA by the en-
zyme phytanoyl-CoA 2-hydroxylase, which
belongs to the family of 2-oxoglutarate-dependent oxygenases, the largest known
family of nonheme metal-dependent oxidiz-ing enzymes (see References 105 and 106
for reviews). Conversion of phytanoyl-CoAto 2-hydroxyphytanoyl-CoA is stoichiomet-
rically coupled to the decarboxylation of
2-oxoglutarate into succinate and CO2, after
which one of the oxygen atoms of the dioxy-gen (O2) molecule is incorporated into thecarboxyl group of succinate and the other in
the 2-hydroxy group of 2-hydroxyphytanoyl-
CoA. The primary sequences of a numberof phytanoyl-CoA 2-hydroxylases from
different species have become available inrecent years with little overall sequence
identity. All phytanoyl-CoA 2-hydroxylasescontain the 2-His-1-carboxylate motif and
the RXS motif responsible for the binding
of iron and 2-oxoglutarate, respectively (107,108). Site-directed mutagenesis studies have
established the identity of His-175, Asp-177,and His-264 as the iron-binding ligands in
the human phytanoyl-CoA 2-hydroxylase
(109).All hydroxylases identified so far con-
tain PTS2 sequences except for the C. ele-
gans 2-hydroxylase, which contains a PTS1-
like sequence (-RSNL) in accordance withthe absence of the PTS2 pathway in C. ele-
gans (42). The next enzyme in the pathway,i.e., 2-hydroxyphytanoyl-CoA lyase, converts2-hydroxyphytanoyl-CoA into pristanal and
formyl-CoA, is a peroxisomal matrix en-zyme of four identical 63-kDa subunits,
and contains a PTS1-like sequence (-SNM)(110). At neutral pH, formyl-CoA is split
into formate and free CoA nonenzymatically
www.annualreviews.org Biochemistry of Peroxisomes 311
-
7/30/2019 New Per Ox i Some Complete
18/41
Figure 5
Schematic
illustration andtopology of theenzymes involvedin the phytanic acidalpha-oxidationpathway inmammalianperoxisomes.
(111). Pristanal is converted into pristanic
acid via a still poorly defined peroxisomalaldehyde dehydrogenase (112). Finally, pris-
tanic acid is activated to pristanoyl-CoA,probably via the enzyme VLCS (104, 113).
Pristanoyl-CoA undergoes three cycles of
beta-oxidation in the peroxisome to produce4,8-dimethylnonanoyl-CoA, which is then
transported to the mitochondria for full ox-idation (68). Recent studies have shown that
peroxisomes also catalyze the oxidation of 2-
hydroxy FAs, which are first activated to the
respective CoA esters, followed by cleavageinto the corresponding aldehyde and formyl-
CoA by 2-hydroxyphytanoyl-CoA lyase (114)
Glyoxylate Metabolism
In humans, the enzyme alanine:glyoxylate
aminotransferase (AGT) is exclusively ex-pressed in liver peroxisomes and converts gly-
oxylate generated in peroxisomes into glycine
312 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
19/41
using alanine as the primary amino group
donor. This prevents the conversion of gly-oxylate into the toxic metabolite oxalate,
which can be catalyzed by various dehydro-genases and oxidases, including lactate dehy-
drogenase. In the case of AGT deficiency, asin patients with hyperoxaluria type 1, glyoxy-
late accumulates and is converted into ox-alate, which precipitates in the liver as wellas in other organs, including the kidneys, ul-
timately causing kidney failure and loss. It hasbeen postulated that glyoxylate, which accu-
mulates in peroxisomes, diffuses across theperoxisomal membrane and is converted into
oxalate by cytosolic lactate dehydrogenase.
Such a scenario is unlikely, however, becauseunder these conditions the cytosolic enzyme
glyoxylate reductase is expected to convert cy-
tosolic glyoxylate to glycolate. Therefore, itis more likely that glyoxylate, which accumu-lates in peroxisomes, is converted into oxalate
by peroxisomal enzymes, including glycolate
oxidase and lactate dehydrogenase (115). In-terestingly, hyperoxaluria type 1 may not only
be caused by a functional deficiency of per-oxisomal AGT but also by mislocalization of
AGT to mitochondria (116). Under the lat-ter conditions, the AGT, mislocalized to mi-
tochondria, is catalytically active, but glyoxy-
late generated in peroxisomes cannot reachthe AGT in mitochondria and instead is con-
verted into oxalate, giving rise to hyperox-aluria type 1 (9).
Amino Acid Catabolism
Mammalian peroxisomes contain d-aminoacid oxidase, which oxidizes the d-isomers
of neutral and basic amino acids, as well asd-aspartate oxidase (117), which oxidizes the
d-isomers of acidic amino acids. The oxida-tion of amino acids by both enzymes yieldsthe corresponding keto acids, ammonia, and
hydrogen peroxide. Peroxisomes are also in-volved in the oxidation of some l-amino acids,
e.g., l-lysine, which may be degraded to l-2-amino adipic acid either via the saccha-
ropine pathway or via the l-pipecolate path-
way. l-pipecolate oxidase, which oxidizes l-pipecolate to 1-piperideine-6-carboxylate,
is a peroxisomal enzyme identified in human
(118) and monkey liver (119), has been pu-rified and cloned (120, 121), and is a typi-
cal PTS1 protein. l-pipecolate accumulatesin tissues and body fluids of patients who lack
peroxisomes, emphasizing the importance ofthe l-pipecolate pathway in humans. Lysine,
hydroxylysine, and tryptophan can be con-
verted to glutaryl-CoA, for which a perox-isomal glutaryl-CoA oxidase has been de-
scribed in rat and man. However, because theglutaryl-CoA oxidase activity copurifies with
palmitoyl-CoA oxidase activity (73), the ex-
istence of a separate glutaryl-CoA oxidase isquestionable.
Pentose Phosphate PathwayIn the pentose phosphate pathway, NADPH
is generated when glucose 6-phosphate is ox-idized to ribose-5-phosphate. Although the
pentose phosphate pathway has always beenassumed to be cytosolic, approximately 10%
of the total activity of the two pentose phos-phatepathway enzymes, glucose-6-phosphate
dehydrogenase and 6-phosphogluconate de-
hydrogenase, is peroxisomal (122). It isproposed that these two enzymes provide
intraperoxisomal NADPH as needed, for ex-ample, for the 2,4-dienoyl-CoA reductase
reaction. As discussed below, peroxisomes
also contain a different system that pro-vides intraperoxisomal NADPH, i.e., via the
2-oxoglutarate/isocitrate NADP(H) shuttle.Because peroxisomes lack any of the sub-
sequent enzymes of the pentose phosphatepathway, the intraperoxisomal generation of
NADPH by the glucose 6-phosphate and 6-
phosphogluconate dehydrogenases would re-quire import of glucose 6-phosphate and ex-
port of ribulose 5-phosphate.
Polyamine Oxidation
Spermine (SPM) and spermidine (SPD) arerequired for numerous fundamentally im-
portant cellular processes, and the levels of
www.annualreviews.org Biochemistry of Peroxisomes 313
-
7/30/2019 New Per Ox i Some Complete
20/41
these polyamines are under tight control.
The main mechanism by which spermine andspermidine are degraded involves transforma-
tion of SPM and SPD into N1-acetyl-SPMand N1-acetyl-SPD by the cytosolic enzyme
acetyl-CoA:SPD/SPM N1-acetyltransferase,followed by oxidation of the N1-acetylated
polyamines by a peroxisomal N1
-acetylatedpolyamine oxidase. This enzyme convertsN1-
acetyl-SPM to SPD and 3-acetamidopropanal
and N1-acetyl-SPD to putrescine and 3-acetamidopropanal. The same enzyme also
oxidizes SPM to SPD and 3-aminopropanal,although very inefficiently. Most cells also
contain a cytosolic spermine oxidase, which
oxidizes SPM to SPD and 3-aminopropanalbut does not react with N1-acetyl-SPM and
N1-acetyl-SPD. The mouse, bovine, and hu-
man peroxisomal N1
-acetylated polyamineoxidases (123, 124) all have a typical PTS1sequence (Table 1). The fate of the prod-
ucts of the peroxisomal polyamine oxidase re-
action is not clear. It may well be that the3-acetamidopropanal is further metabolized
within peroxisomes because peroxisomes har-bor both aldehyde (125) and alcohol dehydro-
genase (126) activities.
Miscellaneous Peroxisomal EnzymeActivities
Peroxisomes have been said to contain a num-ber of different enzyme activities for which
the responsible enzymes and genes have re-
mained unknown as well as proteins with un-known functions (Table 1). Indeed, rat liver
peroxisomes contain a clofibrate-induciblealcohol:NAD+ oxidoreductase activity, with
tetradecanol showing the highest catalytic ef-ficiency, i.e. Vmax/Km (126). Furthermore, a
clofibrate-inducible aldehyde dehydrogenaseactivity was identified in rat liver peroxisomes
with nonanal as the substrate with the high-
est catalytic efficiency (125). Peroxisomes alsocontain pristanal dehydrogenase activity (112)
and retinal reductase activity (4). As discussedabove, the peroxisomal 2,4-dienoyl-CoA re-
ductase is responsible for the latter activ-
ity (4). Peroxisomes also contain a trans-2-enoyl-CoA reductase, which may play a role
in chain elongation (127). Because of the ob-
servation that phytol is converted into phy-tanic acid via the reduction of phytenoyl-CoA
into phytanoyl-CoA, it was recently hypothe-sized that the enoyl-CoA reductase identified
by Das et al. (127) may mediate this reductionstep (128).Recent studies have shown that one or
more members of the nudix hydrolase familyare present in peroxisomes (3). One of these
enzymes, called NUDT7, catalyzes the hy-drolysis of CoA and its derivatives, and its
function may be to eliminate oxidized CoA
from peroxisomes and/or to regulate the lev-els of CoASH and acyl-CoAs in this organelle
in response to metabolic demands. Recently
we performed proteomic analysis of mouseperoxisomes and identified several new per-oxisomal proteins, including a novel nudix
hydrolase designated RP2p and encoded by
the D7RP2e gene. RP2p is a CoA diphos-phatase with activity toward CoASH, oxidized
CoASH, and a wide range of acyl-CoA esters(R. Ofman and R.J.A. Wanders, submitted for
publication). Finally peroxisomes also containNAD-linked glycerol-3-phosphate dehydro-
genase activity (122, 129), which may play a
role in the provision of DHAP for the DHA-PAT reaction (see Figure 1b).
Isoprenoid and CholesterolMetabolism
Many studies performed from the 1950s to the
1980s showed that eight of the nine enzymesof the first part of the isoprenoid biosyn-
thesis pathway, involved in the conversion ofacetyl-CoA to farnesyl pyrophosphate, are cy-
tosolic (130). The exception is 3-hydroxy-3-methylglutaryl-CoA reductase, which hadbeen localized to the endoplasmic reticulum
(ER), as are the enzymes involved specificallyin cholesterol synthesis. Since 1985, however
a series of reports have claimed that many ofthe enzymes (or the reactions they catalyze)
of the first part of the pathway are partly
314 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
21/41
mainly, or even exclusively located in perox-
isomes (131). Moreover, several enzymes in-volved in cholesterol synthesis were reported
to be colocalized in peroxisomes and in theER. This has led to the rather generally ac-
cepted view that peroxisomes would be di-rectly involved in isoprenoid and cholesterol
biosynthesis.Different observations have been used tosupport the claim that peroxisomes would be
involved in isoprenoid/cholesterol biosynthe-sis. Among these arethe decreasedactivities of
several isoprenoid biosynthetic enzymes ob-served in postmortem liver homogenates of
patients, whosuffered from a fatal peroxisome
biogenesis disorder (PBD) and thus lackedfunctional peroxisomes (see References 131
and132for reviews). Recent studies, however,
showed that these decreased activities resultfrom inactivation owing to the bad conditionand/or preservation of the livers rather than
from mislocalization to the cytosol (17). This
corresponds to the finding of normal activi-ties and protein levels of these enzymes in cul-
tured primary skin fibroblasts of PBDpatientsand in liver of the pex5 knockout mouse (18,
133), which constitutes a well-defined modelfor human PBDs (134).
Conflicting data have been published on
the de novo cholesterol synthesis rate inperoxisome-deficient fibroblasts and CHO
cells. Although a few groups reported de-creased rates (135137), others found normal
or even increased rates in such cells (138140),indicating that the loss of peroxisomes per se
does not affect the enzyme activities or de
novo cholesterol biosynthesis.Also, with respect to the subcellular local-
ization of isoprenoid biosynthetic enzymes,conflicting data have been published indicat-
ing either a cytosolic or a peroxisomal local-ization of the proteins. It should be noted,
however, that in most cases the claim of a per-
oxisomal (co)localization was based on (a) thefinding of only (very) low amounts of pro-
teins in peroxisomal fractions obtained aftersubcellular fractionation of rat liver tissue, (b)
immunocytochemicalstudies using antiseraof
PBD: peroxisombiogenesis disorde
undefined specificity, and/or (c) the results of
overexpression studies with tagged proteinsor(portions of) proteins fused to reporter pro-
teins in cell lines. As discussed above, the lat-ter studies can be informative but may be full
of pitfalls and can only support, but neverfully replace, studies aimed at determining
the subcellular localization under physiolog-ical conditions. Indeed, the subcellular lo-calization under physiological conditions and
after overexpression of the three human iso-prenoid biosynthetic enzymes mevalonate ki-
nase, phosphomevalonate kinase, and meval-onate pyrophosphate decarboxylase, which
were previously claimed to be predominantly
peroxisomal, was reinvestigated in great detailusinga variety of biochemical andmicroscopi-
caltechniques.The results of these studies un-
ambiguously pointed to an exclusive cytosoliclocalization of these enzymes with no indica-tion of even a partial peroxisomal localization
(1618).
When combining all available data withemphasis on studies of the subcellular lo-
calization of authentic, nonengineered pro-teins under physiological conditions, the con-
clusion must be that there is little, if any,evidence for a direct peroxisomal involve-
ment in the biosynthesis of isoprenoids and
cholesterol.
BIOCHEMISTRY OF HUMANPEROXISOMAL DISORDERS
The importance of peroxisomes for normal
mammalian development and growth is un-derlined by the existence of a group of in-
herited diseases in humans, the peroxisomaldisorders, which can be classified into two
groups, including(a)thePBDsand(b)thesin-gle peroxisomal enzyme deficiencies. The fa-tal cerebro-hepato-renal syndrome, in short
Zellweger syndrome (ZS), is the prototypeof the first group and is characterized by
the complete absence of peroxisomes. Theunderlying basis for the inability to synthe-
size peroxisomes in ZS has been resolved in
www.annualreviews.org Biochemistry of Peroxisomes 315
-
7/30/2019 New Per Ox i Some Complete
22/41
recent years and involves mutations in at least
12 differentPEXgenes (141143).The absence of peroxisomes has major
consequences for most of the metabolic path-ways in which peroxisomes are involved.
This is evident from the biochemical ab-berrations observed in ZS patients; these
abberrations range from the accumulationof substrates normally handled by peroxi-somes, e.g., VLCFAs, pristanic acid, phytanic
acid, DHCA, THCA, and pipecolic acid, toa shortage of end products of peroxisomal
metabolism, e.g., plasmalogens, cholic andchenodeoxycholic acid, and docosahexaenoic
acid (see Table 2). From these and other ob-
servations, it hasbecome clear that theseques-tration of (or parts of ) certain metabolic path-
ways in peroxisomes is essential for efficient
substrate channeling and to protect the cellagainst toxic metabolites generated in perox-isomes, e.g., reactive oxygen species, reactive
nitrogen species, and glyoxylate. The abnor-
malities observed in ZS patients are causedby the deficiency of most, but not all, per-
oxisomal enzymes destined for peroxisomes.Indeed, most peroxisomal enzymes are unsta-
ble in the cytosol and are rapidly degraded,whereas a few peroxisomal enzymes, such as
catalase (144) and alanine:glyoxylate amino-
transferase (145), are assembled correctly intoactive multimers (tetramer and dimer, respec-
tively) and are stable in the cytosol.Most peroxisomal disorders belong to
group two, which can be subdivided fur-ther into distinct subgroups, depending
upon which peroxisomal function is impaired
(Table 2). Virtually all peroxisomal enzymedeficiencies are associated with severe clinical
aberrations. Remarkably, defects in enzymeswithin the same metabolic pathway may re-
sult in different phenotypes (Table 2). Thisis especially true for the disorders of perox-
isomal beta-oxidation (Table 2). For exam-
ple, patients with DBP deficiency are severelyaffected with clinical signs and symptoms re-
sembling those observed in ZS patients. In pa-tients with DBP deficiency, the peroxisomal
beta-oxidation of all major substrates is im-
paired, resulting in the accumulation of VL-CFAs, pristanic acid, DHCA, and THCA in
tissues and plasma (85, 146). Conversely, pa-
tients with AMACR deficiency show a mildclinical phenotype resembling Refsum dis-
ease (see below) and only accumulate pris-tanic acid, DHCA, and THCA (147, 148)
Another example is X-linked adrenoleukodys-trophy, which affects boys who develop nor-
mally for the first few years of life and then
rapidly deteriorate, followed by early deathIn X-linked adrenoleukodystrophy, only VL-
CFAs accumulate (68, 149).Other single peroxisomal enzyme de
ficiencies are rhizomelic chondrodysplasiapunctata type 2 and 3, caused by mutations
in GNPAT and AGPS, respectively, which
encode the peroxisomal enzymes DHAPAT
and ADHAPS, respectively. Patients affectedby these deficiencies show markedly loweredplasmalogen levels, which is in line with the
notion that both enzymes play an indispens-able role in ether-phospholipid biosynthesis
(52). Refsum disease also belongs to group 2
and is caused by mutations in the gene en-coding phytanoyl-CoA hydroxylase; as a re-
sult, phytanic acid gradually accumulates andreaches toxic levels later in life (150, 151). An-
other disorder belonging to group 2 is hyper-
oxaluria type 1, caused by mutations in thealanine:glyoxylate aminotransferase encoding
gene (AGXT). When glyoxylate accumulatesin peroxisomes, oxalate is formed, which pre-
cipitates as calcium oxalate in tissues, includ-ing the kidney. This explains the patients loss
of kidney function over time. Acatalasaemia isthe last single peroxisomal enzyme deficiency
caused by mutations in the catalase-encoding
gene, and is associated with an increased ten-dency to develop oral gangrene in otherwise
asymptomatic patients (152).
MOUSE MODELS FORPEROXISOMAL DISORDERS
Because the number of patients with spe-cific peroxisomal defects is rather limited and
the majority of defects lead to early death
316 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
23/41
Table2
Biochemistry
ofhumanperoxisomaldisordersa
Biochemicalabnormalities
Groups
Pe
roxisomaldisorder
Mutantgen
e
VLCFA
PRIS
PHYT
D
/THCA
PL
Clinicalsign
sandsymptoms
Group1
(peroxisome
biogenesis
defects)
Zellwegerspectrum
disorders(ZS,NALD,
IRD)
PEX1,2,3,5,
6,10,1
3,14,
16,1
9,26
b
b
ZS,NALD,andIRD
representaspectrum
ofdiseaseseveritywithZSbeingthemost
andIRDtheleasts
everedisorder.
CommontoZS,N
ALD,andIRDareliver
disease,variablene
uro-developmental
delay,retinopathy,andperceptivedeafness.
ZSpatientsareusu
allyhypotonicfrom
birthanddiebeforeoneyearofage,
whereasNALDpatientsshowneonatal
onsethypotoniaan
dseizures,andthey
haveprogressivewhitematterdisease,
usuallydyinginlateinfancy.IRDpatients
maysurvivebeyondinfancy,andsomemay
evenreachadulthood
RCDPtype1
PEX7
N
N
b
N
Patientshaveadisproportionallyshort
statureprimarilyaf
fectingtheproximal
partsoftheextremities.Othersymptoms
includetypicalfacialabnormalities,
congenitalcontractures,ocular
aberrations,severe
growthdeficiency,and
mentalretardation
Group2(single
peroxisomal
enzyme
deficiencies)
X-linked
adrenoleukodystrophy
(X-ALD)
ABCD1
N
N
N
N
Twomajorforms,in
cludingchildhood
cerebraladrenoleukodystrophy(CCALD)
andadrenomyeloneuropathy(AMN);in
thesevereform(CCALD),normal
developmentuntilsixyearsofage,
followedbyrapidd
eteriorationanddeath
withintwoyears
(Continued)
www.annualreviews.org Biochemistry of Peroxisomes 317
-
7/30/2019 New Per Ox i Some Complete
24/41
Table2
(Continued)
Biochemicalabnormalities
Groups
Pe
roxisomaldisorder
Mutantgen
e
VLCFA
PRIS
PHYT
D
/THCA
PL
Clinicalsign
sandsymptoms
Acyl-CoAoxidase
deficiency(ACOX1
deficiency)
ACOX1
N
N
N
N
Hypotonia,earlyonsetseizures,hearing
loss,retinopathy,neurological
abnormalities
d-Bifunctionalproteinde-
ficiency/multifunctional
pro
tein2deficiency
(DBP/MFP2deficiency)
HSD17B4
b
b
N
Craniofacialabnorm
alities;neurological
disturbances;Zellw
eger-likephenotype,
includingneuronal
migrationdefect
2-M
ethyl-acyl-CoA
rac
emasedeficiency
(AMACRdeficiency)
AMACR
N
b
b
N
Slow,progressivelossofvision;neurological
deterioration;inso
mepatientsmarked
hepatopathy
RCDP2(DHAPAT
deficiency)
GNPAT
N
N
N
N
Severegrowthretardation,mental
retardation,rhizom
elia,earlydeath
RCDP3(ADHAPS
deficiency)
AGPS
N
N
N
N
Severegrowthretardation,mental
retardation,rhizom
elia,earlydeath
Refsumdisease
(ph
ytanoyl-CoA
hydroxylasedeficiency)
PAHX/PHYH
N
N
N
N
Lossofvision,cereb
ellarataxia,anosmia,
ichtyosis,cardiacproblems
Hyp
eroxaluriatype1
(AGTdeficiency)
AGXT
N
N
N
N
N
Progressivelossofk
idneyfunction
Acatalasaemia
CAT
N
N
N
N
N
Increasedtendencytodeveloporalgangrene
aAbbreviations:ZS,Zellwege
rsyndrome;NALD,neonataladrenoleukod
ystrophy;IRD,infantileRefsumdisease;RC
DP,rhizomelicchondrodysplasiapunctata;X-ALD,X-linked
adrenoleukodystrophy;VLCFA,very-long-chainFAs;PRIS,pristanicacid;PHYT,phytanicacid;D/THCA,di-andt
rihydroxycholestanoicacid;PL,plasmalogens;N,normal;,elevated;,
decreased.
bLevelsmayvaryfromnormaltoelevatedbecausephytanicacidandpristanicacidarederivedfromexogenous(dietary)sourcesonly.
318 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
25/41
most biochemical studies on these defects
have been done on cells of such patients, inparticular primary skin fibroblasts. Although
valuable and informative, these studies do notshow how and why a given peroxisomal dys-
function leads to the specific pathophysiologyassociated with these defects. To obtain more
insight into these pathophysiological conse-quences,anumberofmousemodelshavebeengenerated; the biochemical and phenotypical
characteristics of these models are summa-rized in Table 3. All of these mice were gen-
erated through targeted gene disruption.Most mouse models, for which currently a
human peroxisomal disorder is known, show
biochemical and phenotypical defects similartothoseobservedinthecorrespondinghuman
disorders. Moreover, studies on these mouse
models have led to the identification of ad-ditional defects that could not be readily in-
vestigated in patients or their cells, including
ossification and neuronal migration defects.
A few mouse knockouts have been gener-ated that create peroxisomal enzyme deficien-
cies for which no human peroxisomal disorderhas been identified (see Table 3). The bio-
chemicaland phenotypical characterization ofthese mouse models may aid in the recogni-
tion of their possible human counterparts. In-
terestingly, it appears that, at least in mice, theabsence of certain peroxisomal enzymes in-
cluding l-bifunctional enzyme (80), thiolase-B (153), and catalase (154) does not result in
noticeable biochemical and phenotypical ab-normalities, making the specific physiologi-
cal function of these enzymes difficult to dis-
cern. It should be noted, however, that suchenzymes maybe required only for certain spe-
cific conditions, as exemplified by the Scp2
(/) mouse. In this mouse model, severe
biochemical and phenotypical defects becameapparentonlyafterfeedingitthephytanicacid
precursor phytol, leading to the accumulation
of pristanic acid. From this observation andadditional findings in the Amacr(/) mouse
(155), one may predict that the possible hu-man counterpart of SCP deficiency presents
onlylaterinlife,asisthecasewithRefsumdis-
ease, which presents only after gradual accu-mulation of phytanic acid to toxic levels over
time.
Another aspect that should be mentionedis the short life span of mice in comparison to
humans, which maynot allow enoughtime forthe development of certain phenotypical ab-
normalities. One example is the observationthatAbcd1 (/) mice, a mouse model for X-
linked adrenoleukodystrophy (X-ALD), only
develop mild neurological and behavioral ab-normalities later in life (> 15 months of age),
and these abnormalities resemble those ob-served in adrenomyeloneuropathy, a milder
variant of X-ALD (156).The generation of additional mouse mod-
els for selected peroxisomal proteins has been
reported forwhichno detailed information on
the biochemical and phenotypical character-istics is available yet. These include mice withtargeted disruptions of the genes encoding
phytanoyl-CoA hydroxylase (a model for Ref-sum disease), peroxisomal ABC proteins, i.e.,
the 70-kDa peroxisomal membrane protein
(PMP70), ALD-related protein (ALDRP),PMP70-related protein (PMP70R) (no hu-
man diseases known), and alanine:glyoxylateaminotransferase (a model for hyperoxaluria
type 1).
PEROXISOMAL METABOLITETRANSPORT
Correct execution of the many metabolic
functions of peroxisomes requires the trans-port of a large variety of metabolites across
the peroxisomal membrane. In recent years,much hasbeen learned about the permeability
properties of peroxisomes as discussed below.
Permeability Properties ofPeroxisomes
Because after isolation mammalian peroxi-somes are freely permeable to low-molecular-
weight compounds and peroxisomal enzymesdo not show structure-linked latency, it has
long been assumed that the peroxisomal
www.annualreviews.org Biochemistry of Peroxisomes 319
-
7/30/2019 New Per Ox i Some Complete
26/41
Table3
Mousemodelsofperoxisomebiogenesisandperoxisomefunctiona
Disrupted
gene
Deficient
(enzyme)protein
Corresponding
humandisease
Biochemicalphenotype
VLC
FA
PRIS
PHYT
D/THCA
PL
Clinicalcharacteristics
References
Pex2
Pex2
Zellweger
spectrum
disorder
(ZS/NALD/IRD)
Intrauterinegrowthretardation,
severehypotonia,neonatald
eath,
delayedneuronalmigration
inCNS,
cerebellarabnormalitieswith
reducedPurkinjecelldevelo
pment
(192,193)
Pex5
Pex5
Zellweger
spectrum
disorder
(ZS/NALD/IRD)
Lowbirthweight,hypotonia,poor
feeding,neuronalmigration
defect,
neonataldeath
(134,194)
Pex13
Pex13
Zellweger
spectrum
disorder
(ZS/NALD/IRD)
Lowbirthweight,hypotonia,poor
feeding,neuronalmigration
defect,
neonataldeath
(195)
Pex7
Pex7
RCDPtype1
N
N
N
Intrauterinegrowthretardation,
severehypotonia,delayedossification
ofdistalboneelements,dwa
rfism,
delayedneuronalmigration
(196)
Pex11
Pex11
N
N
N
N
N
Nophenotypicabnormalities
(197)
Pex11
Pex11
Zellweger
spectrum
disorder
(ZS/NALD/IRD)
N
N
N
N
N
Intrauterinegrowthretardation,
hypotonia,developmentaldelay,
neonataldeath,impairedneuronal
migration
(198)
Gnpat
Dhapat
RCDPtype2
N
N
N
N
Intrauterinegrowthretardation,
hypotonia,maleinfertility,d
efectsin
eyedevelopment,cataract,o
ptic
nervehypoplasia,prenataldeathof
Dhapat(/)embryos
(199)
Acox1
Acox1
Acyl-CoAoxidase
deficiency
N
N
N
N
Viable,butinfertile;retarded
postnatalgrowth;microvesicular
steatosis;focalcelldeath;
inflammatoryreactions;livertumors
atlaterage(>15months)
(200)
320 WandersWaterham
-
7/30/2019 New Per Ox i Some Complete
27/41
Hsd17B4
Dbp/Mfp2
d-Bifunctional
proteindeficiency
N
Normalbirthweight,dramat
icgrowth
retardation,upto30%diebefore
postnatalday12,maleinfertility,no
neuronalmigrationdefect
(86)
Scp2
Peroxisomal
thiolase2
(Scpx)
N
N
Nophenotypicabnormalities,phytol
feedinginducesweightloss,
neurologicalabnormalities,
andearly
deathwithinthreeweeksof
birth
(94)
Abcd1
Aldp
Adrenomyel-
oneuropathy
N
N
N
N
Noapparentphenotype;how
ever,
beyondage15months,late-onset
neurologicalandbehavioral
abnormalities,axonallossin
the
spinalcord,andslowernerv
e
conduction
(156,
201203)
Ehhadh
Lbp/Mfp1
N
N
N
N
N
Nophenotypicabnormalities
(204)
Slc27a2
Vlcs
N
N
N
N
N
Nophenotypicabnormalities
(176)
Amacr
Amacr
AMACR
deficiency
N
N-
N
Nophenotypicabnormalities,
intolerancetophytolwithliver
diseaseandearlydeath
(155)
mThB
ThiolaseB
N
N
N
N
N
Nophenotypicabnormalities
(153)
Cat
Catalase
Acatalasemia
N
N
N
N
N
Nophenotypicabnormalitiesexcept
forincreasedsusceptibilityto
trauma-induceddysfunction
ofbrain
mitochondria
(154)
aSeeTable2forabbreviations.
www.annualreviews.org Biochemistry of Peroxisomes 321
-
7/30/2019 New Per Ox i Some Complete
28/41
membrane does not constitute a permeability
barrier to small molecules, at least in mam-mals. More recent studies, notably in the yeastS. cerevisiae and partly confirmed in mam-malian cells, however, revealed that in vivo
the peroxisomal membrane is impermeable tosmall metabolites, which implies the existence
of peroxisomal metabolite carriers. Hence, itseems plausible that the in vitro permeabilityis due to the disruption of protein/membrane
structures as a consequence of the cell frac-tionation methods used for their isolation. It
should be noted that structure-linked latencyof peroxisomal enzymes has been observed
in other members of the microbody family,
including glyoxysomes and glycosomes. Onthe basis of pulse-labeling experiments in Try-
panosoma brucei, it was concluded that the gly-
cosomal membrane is poorly permeable toglycolytic intermediates with the exception ofglycerol-3-phosphate, which was postulated
to be transported via a specific translocator
(157). Recent studies by Antonenkov et al.(158, 159) have shown that under properly
controlled conditions several peroxisomal en-zymes do show structure-linked latency in
peroxisomes from rat liver.
The Intraperoxisomal pHOne of the first indications that the perox-
isomal membrane may form a closed struc-ture in vivo was the demonstration of a pH
gradient across the membrane in yeast, which
could be dissipated by uncouplers (160).Morerecently, a peroxisomal pH gradient was also
shown in human cells (161). It remains un-clear whether this gradient is due to an ac-
tive proton-translocating protein, the conse-quence of a high intraperoxisomal metabolic
activity, or both. Moreover, there is no agree-ment concerning the orientation of the pro-ton gradient because some groups reported a
lower (162), whereas others found a higheror similar intraperoxisomal pH in compari-
son to the cytosolic pH (163), despite the factthat similar methodologies and cell systems
were used. This also makes it difficult to de-
termine whether theproton gradient is used asa driving force for metabolite transport, e.g.
FA transport (163), or is a mere consequence
of metabolite transport (164). Further insightinto a possible physiological function of a per-
oxisomal proton gradient may come from invitro studies with reconstituted peroxisoma
membrane proteins aimed at determining theproperties, characteristics, and requirements
of peroxisomal metabolite carriers and shut-
tle systems,which were identified recently.
Peroxisomal ABC Transporters
Mamma