1890Journal of Lipid Research
Volume 41, 2000
Subcellular localization and physiological role of
Sacha Ferdinandusse,* Simone Denis,* Lodewijk IJlst,* Georges Dacremont,
and Ronald J. A. Wanders
Departments of Clinical Chemistry* and Pediatrics,
University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; and Department of Pediatrics,
of Ghent, 9000 Ghent, Belgium
Hans R. Waterham,
Emma Children’s Hospital, Academic Medical Center,
role in the
fatty acid derivatives because it catalyzes the conversion of
)-methyl-branched-chain fatty acyl-CoAs to their
)-stereoisomers. Only stereoisomers with the 2-methyl
group in the (
)-configuration can be degraded via
oxidation. Patients with a deficiency of
racemase accumulate in their plasma pristanic acid and the
bile acid intermediates di- and trihydroxycholestanoic acid,
which are all substrates of the peroxisomal
tem. Subcellular fractionation experiments, however, re-
vealed that both in humans and rats
mase is bimodally distributed to both the peroxisome and
the mitochondrion. Our findings show that the peroxisomal
and mitochondrial enzymes are produced from the same
gene and that, as a consequence, the bimodal distribution
pattern must be the result of differential targeting of the
same gene product. In addition, we investigated the physio-
logical role of the enzyme in the mitochondrion.
vitro studies with purified heterologously expressed protein
and in vivo studies in fibroblasts of patients with an
ylacyl-CoA racemase deficiency revealed that the mitochon-
drial enzyme plays a crucial role in the mitochondrial
oxidation of the breakdown products of pristanic acid
by converting (2
,6)-dimethylheptanoyl-CoA to its (
—Ferdinandusse, S., S. Denis, L. IJlst, G. Dacre-
mont, H. R. Waterham, and R. J. A. Wanders.
localization and physiological role of
J. Lipid Res.
-Methylacyl-CoA racemase plays an important
-oxidation of branched-chain fatty acids and
Supplementary key words
branched-chain fatty acid
Peroxisomes in mammals harbor two distinct pathways
for fatty acid
-oxidation. The first pathway catalyzes the
-oxidation of very long-chain fatty acids, such as C26:0,
and the second pathway catalyzes the
branched-chain fatty acids and fatty acid derivatives, such
as pristanic acid and the bile acid intermediates di- and
trihydroxycholestanoic acid (DHCA
tively). The central role of peroxisomes in the oxidation
and THCA, respec-
of branched-chain fatty acids and fatty acid derivatives is
clearly demonstrated by studies in patients with Zellweger
syndrome, who lack functional peroxisomes. Analysis of
plasma from these patients reveals a series of abnormali-
ties including the accumulation of DHCA, THCA, phy-
tanic acid, and pristanic acid, which is derived from phy-
tanic acid after one cycle of
(1). Previous studies have shown that the peroxisomal
-oxidation system is stereospecific (2–4), because the per-
oxisomal oxidases [branched-chain acyl-coenzyme A (CoA)
oxidase in humans and trihydroxycholestanoyl-CoA (THC-
CoA) oxidase and pristanoyl-CoA oxidase in rat] can handle
only the (
S)-stereoisomer of 2-methyl-branched acyl-CoAs
(2, 3). Because both phytanic acid (3,7,11,15-tetramethyl-
hexadecanoic acid) and pristanic acid (2,6,10,14-tetrameth-
ylpentadecanoic acid) naturally occur as a mixture of two dif-
ferent diastereomers [(2
S ,6R ,10
case of pristanic acid] (5), the (2
needs to be converted to its (
substrate for the peroxisomal
conversion is catalyzed by a racemase called
CoA racemase, which catalyzes the interconversion of a
large variety of (
R)- and (S )-2-methyl-branched-chain fatty
acyl-CoAs (6–9). The same racemase is also essential for
the degradation of DHCA and THCA (7, 9), of which only
R )-stereoisomers are produced via (
tochondrial 27-hydroxylation (10) (Fig. 1).
Studies on the subcellular localization of
CoA racemase revealed that the enzyme activity is not only
localized in peroxisomes but is also present in mitochon-
dria, at least in humans (7, 8). In rat, however, the local-
ization is controversial. Conzelmann and co-workers, who
-oxidation in the peroxisome
R ) and (2
)-stereoisomer to become
R ,6R,10R ) in the
)-pristanic acid first
Abbreviations: DHCA, dihydroxycholestanoic acid; EDTA, ethyl-
enediaminetetraacetic acid; HPLC, high performance liquid chroma-
tography; LCAD, long-chain acyl-CoA dehydrogenase; MBP, maltose-
binding protein; MOPS, morpholinepropane sulfonic acid; THCA, tri-
hydroxycholestanoic acid; THC-CoA, trihydroxycholestanoyl coenzyme A.
To whom correspondence should be addressed.
by guest, on June 5, 2013
1896Journal of Lipid Research
Volume 41, 2000
(S)-stereoisomer, the only stereoisomer that can be ?-oxi-
dized in the mitochondrion (4, 14), and found that this was
indeed the case. Subsequently, we studied whether this is the
true physiological function of the enzyme. We found that fi-
broblasts from patients with an established ?-methylacyl-CoA
racemase deficiency were not able to convert (2R,6)-dimeth-
ylheptanoyl-CoA to its (S)-stereoisomer, which confirms that
?-methylacyl-CoA racemase activity is essential at several steps
in the degradation of pristanic acid to CO2 and H2O in the
peroxisome as well as in the mitochondrion.
To obtain additional in vivo evidence of the role of mi-
tochondrial racemase in the oxidation of 2,6-dimethyl-
heptanoyl-CoA, we performed acylcarnitine analysis in ?-
methylacyl-CoA racemase-deficient patients. These studies
did not reveal accumulation of 2,6-dimethylheptanoyl-
carnitine (data not shown). The reason for this is most
probably that even though half the pristanic acid can
enter the ?-oxidation spiral in these patients, as it natu-
rally occurs as a racemic mixture (see Fig. 2), it cannot
proceed beyond 2,6,10-trimethylundecanoyl-CoA, of which
all methyl groups have the (R)-configuration. For this com-
pound to be further ?-oxidized, the (2R)-methyl group
needs to be converted to the (S)-configuration, which is
most likely also catalyzed by ?-methylacyl-CoA racemase
(see Fig. 2). This is supported by the finding of 2,6,10-
trimethylundecanoyl-carnitine in the plasma of one of the
We thank R. Ofman, F. M. Vaz, and C. J. Dekker for technical
assistance. This work was supported by the Princess Beatrix
Fund (The Hague, The Netherlands).
Manuscript received 3 March 2000 and in revised form 19 May 2000.
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