Báo cáo khoa học: Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence pptx

Van Veldhoven Afdeling Farmacologie, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Belgium 3-Methyl-branched fatty acids, as phytanic acid, undergo peroxisomal a-

R E V I E W A R T I C L E

Alpha-oxidation of 3-methyl-substituted fatty acids

and its thiamine dependence

Minne Casteels, Veerle Foulon, Guy P Mannaerts and Paul P Van Veldhoven

Afdeling Farmacologie, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Belgium

3-Methyl-branched fatty acids, as phytanic acid, undergo

peroxisomal a-oxidation in which they are shortened by 1

carbon atom This process includes four steps: activation,

2-hydroxylation, thiamine pyrophosphate dependent

cleavage and aldehyde dehydrogenation The thiamine

pyrophosphate dependence of the third step is unique in

peroxisomal mammalian enzymology Human pathology

due to a deficient alpha-oxidation is mostly linked to mutations in the gene coding for the second enzyme of the sequence, phytanoyl-CoA hydroxylase

Keywords: alpha-oxidation; thiamine pyrophosphate; per-oxisomes; lyase; Adult Refsum Disease

Introduction

a-Oxidation is the process in which fatty acids are shortened

at the carboxyl-end by one carbon atom For

3-methyl-branched fatty acids, this is the preferred pathway as their

breakdown by b-oxidation is impossible Indeed, the

3-methyl-branch precludes the third step of b-oxidation,

the dehydrogenation step Phytanic acid

(3,7,11,15-tetra-methylhexadecanoic acid) is at present the only established

physiological substrate of a-oxidation in humans [1,2]

Phytanic acid is derived from phytol, the isoprenoid side

chain of chlorophyll As chlorophyll-bound phytol cannot

be metabolized by humans, and free phytol is present only

in minimal quantities in food, the phytanic acid present in

the human body is mostly provided by external sources

(Fig 1) Ruminants ingest large amounts of chlorophyll,

from which phytol is efficiently cleaved off by bacteria in the

gastrointestinal tract Phytol is subsequently taken up and

converted to phytanic acid, which is deposited in fat tissues

and in milk, the major sources of phytanic acid for humans

[2]

Accumulation of phytanic acid is typically seen in Adult

Refsum Disease (ARD) and is due to a deficient

degrada-tion of this exogenous 3-methyl-branched fatty acid [2,3]

Elevated phytanic acid levels can also be seen in peroxisome

biogenesis disorders, in which a defective a-oxidation is only

one of the deficiencies present [4] Degradation of phytanic

acid via x-oxidation, by which a carboxylic acid group is

introduced at the omega end, has also been described [5,6], but appears to be quantitatively less important under physiological conditions Its importance increases when phytanic acid levels in serum are elevated as is seen in ARD [7]

The degradation of phytanic acid via a-oxidation is presently proposed to evolve completely in peroxisomes, some doubts remaining, however, concerning the first (activation) and last (aldehyde dehydrogenation) enzymatic steps

Degradation of 3-methyl-branched fatty acids The classic catabolic pathway by which fatty acids are degraded is b-oxidation and a mitochondrial as well as a peroxisomal b-oxidation pathway is known [8] Very long chain fatty acids, 2-methyl-branched fatty acids, the side chains of bile acid intermediates and eicosanoids are mainly/ exclusively handled by the peroxisomal pathway, whereas short and medium chain fatty acids are oxidized mainly in mitochondria [8]

Phytanic acid and other 3-methyl-branched fatty acids cannot undergo b-oxidation because the 3-methyl-group prevents the formation of a 3-keto substituent in the dehydrogenation step Therefore, 3-methyl-branched fatty acids first undergo a-oxidation In the case of phytanic acid, this results in the generation of 2-methyl-branched pristanic acid (2,6,10,14-tetramethylpentadecanoic acid), which is then shortened to 4,8-dimethylnonanoic acid via peroxi-somal b-oxidation The dimethyl fatty acid is then degraded further via mitochondrial b-oxidation

Peroxisomes, in which most or all steps of the a-oxidation pathway evolve, are subcellular organelles involved in a number of anabolic (e.g plasmalogen synthesis) and catabolic processes, including a- and b-oxidation [8] Peroxisomal enzymes are synthesized on polyribosomes in the cytosol and are post-translationally imported into the peroxisome Therefore, these enzymes contain a series of conserved amino acids or so called peroxisome targeting signals (PTSs) [9] Two classes of these topogenic sequences

Correspondence to M Casteels, Afdeling Farmacologie, Department

of Molecular Cell Biology, Katholieke Universiteit Leuven,

Campus Gasthuisberg, Herestraat 49, B 3000 Leuven, Belgium.

Fax: + 32 16 345699, Tel.: + 32 16 345816,

E-mail: minne.casteels@med.kuleuven.ac.be

Abbreviations: PAHX, phytanoyl-CoA hydroxylase; 2-HPCL,

2-hydroxyphytanoyl-CoA lyase; ARD, Adult Refsum Disease;

PTS, peroxisome targeting signal; TPP, thiamine pyrophosphate.

(Received 15 November 2002, revised 15 February 2003,

accepted 21 February 2003)

have been described: PTS1, a carboxy-terminal tripeptide,

and PTS2, an amino-terminal nonapeptide [9] A defect in

the PTS-receptors or other components of the import

machinery results in a generalized peroxisome biogenesis

disorder [4]

a-Oxidation of 3-methyl-branched fatty acids has already

been studied in the sixties and seventies, but only in the last

decade have most aspects of a-oxidation been unravelled [8]

For the study of this pathway both the natural substrate

phytanic acid, racemic at carbon 3, and the synthetic

(3-R,S)-methylhexadecanoic and

(3-R,S)-methylheptadeca-noic acids, have been used It has been shown that the

synthetic 3-methyl-branched fatty acids are metabolized in

the same way as phytanic acid [10], and can validly be used

as substitutes for the latter substrate when studying

a-oxidation A major breakthrough in a-oxidation research

was Poulos’ finding that in fibroblasts a-oxidation of

3-methyl-branched fatty acids generates not only CO2, as

was generally believed, but also formate [11] Up till then

only CO2had been measured as an end product, and major

discrepancies existed between oxidation rates obtained in

intact cells (isolated hepatocytes, confluent fibroblasts),

permeabilized hepatocytes and broken cell systems (liver

homogenates, subcellular fractions) [8] Subsequent

meas-urements of formate (plus formyl-CoA, see below) and CO2

resolved the discrepancies between intact and

permeabi-lized/broken systems and allowed for the dissection of the

a-oxidation process Our present knowledge of the

enzy-matic sequence is shown in Fig 2

In a first step the 3-methyl-branched fatty acid is activated

to the corresponding CoA-ester by an acyl-CoA synthetase

which is most probably present in the peroxisomal

membrane It is not yet clear which synthetase is responsible for the activation step: a nonspecific long chain fatty acyl-CoA synthetase [12], a specific phytanoyl-acyl-CoA synthetase [13] or a very long chain fatty acyl-CoA synthetase [14] The second step is responsible for the iron dependence of the pathway [15], which had been described by several authors in the past but was regarded as doubtful concerning its physiological relevance [16,17] In this step the 3-methylacyl-CoA is hydroxylated in position 2 by a dioxygenase, which is dependent on molecular O2, iron, 2-oxoglutarate, ascorbate, ATP/GTP and Mg2+ [18–21] This dioxygenase, named phytanoyl-CoA hydroxylase (PAHX), contains a PTS2-signal and is present in the peroxisomal matrix [22,23] The product of the reaction

Fig 1 Chemical structures of chlorophyll, phytol and phytanic acid

(3,7,11,15-tetramethylhexadecanoic acid).

Fig 2 a-Oxidation of 3-methyl-branched fatty acids The scheme represents the a-oxidation pathway of phytanic acid The numbers indicate the enzymes catalysing the different steps: (1) acyl-CoA syn-thetase; (2) phytanoyl-CoA hydroxylase (PAHX); (3) 2-hydroxy-phytanoyl-CoA lyase (2-HPCL); (4) aldehyde dehydrogenase; and (5) formyl-CoA hydrolase.

catalysed by PAHX is a 2-hydroxy-3-methylacyl-CoA, or, if

phytanic acid is the substrate, 2-hydroxyphytanoyl-CoA

The PAHX gene is located on chromosome 10 [22], and

mutations of this gene are probably the most frequent cause

of ARD [22–25] Structure-function analysis of PAHX

further revealed that at least four different types of

mutations can cause loss of enzyme activity [25]

In the third step, 2-hydroxy-3-methylacyl-CoA is cleaved

in the peroxisomal matrix [26,27] by

2-hydroxyphytanoyl-CoA lyase (2-HPCL), which uses thiamine pyrophosphate

(TPP) as cofactor [26] Products of this reaction are

formyl-CoA [28] and a 2-methyl-branched fatty aldehyde (pristanal

when 2-hydroxyphytanoyl-CoA is cleaved) [29,30], both of

which had been identified before the discovery of the lyase

(see below)

The 2-methyl-branched fatty aldehyde is subsequently

dehydrogenated by an NAD+-dependent aldehyde

dehy-drogenase to a 2-methyl-branched fatty acid (pristanic acid

in the case of pristanal), which can be activated to the

corresponding acyl-CoA ester This CoA-ester can then

enter the peroxisomal b-oxidation sequence The 2-methyl

aldehyde dehydrogenase activity is located in the

peroxi-somal matrixaccording to Croes et al [29] and in the

endoplasmic reticulum (microsomes) according to

Verho-even et al [30] It remains at present unclear which aldehyde

dehydrogenase is involved Measurements in Sjo¨gren–

Larsson syndrome (SLS) fibroblasts, the microsomal

alde-hyde dehydrogenase of which is deficient, show only a 30%

decrease in dehydrogenation rates of pristanal [31,32] and

make an exclusive role of a microsomal aldehyde

dehy-drogenase unlikely

The major part of formyl-CoA is enzymatically converted

to formate in peroxisomes [28] It was shown previously [33] that in rats, aminotriazole, known as an inhibitor of catalase, had little effect on the conversion of14C-formate to

CO2(but decreased the rates of a-oxidation by 90%) In rat formate is metabolized by two pathways: the catalase pathway and the tetrahydrofolate pathway, important in one carbon-metabolism [34] The data on aminotriazole indicate that at least in the rat the catalase pathway is of no paramount importance, and suggest that the tetrahydro-folate pathway is quantitatively more important for formate metabolism [33] We studied the conversion of14C-formate

to14CO2in rat and found it to be localized mainly in the cytosolic fraction, and to be stimulated by NAD+[19] No further work on the fate of formate as a product of a-oxidation has been published since Nothing is known

on the export of formate from the peroxisome, but it is supposed that formate, as well as other small organic acids can leak from the peroxisomes [35]

Table 1 gives an overview of the presently known characteristics of the four main enzymes of the a-oxidation pathway

Stereospecificity of the a-oxidation pathway Phytol has two chiral centres, one at carbon 7 and one at carbon 11, both of which are of the R-configuration [41] Non-specific reduction of the double bond in phytol leads

to the production of two diastereoisomers: (3S,7R,11R)-and (3R,7R,11R)-phytanic acid [42] Phytanic acid from all common sources is a mixture of these two

Table 1 Properties of the enzymatic steps/enzymes of the a-oxidation pathway The table gives an overview of the present knowledge of some of the properties of the enzymes involved in the initial degradation of 3-methyl-branched fatty acids in humans See text for details.

Acyl-CoA synthetase

Phytanoyl-CoA hydroxylase (PAHX)

2-Hydroxyphytanoyl-CoA lyase (2-HPCL)

Aldehyde dehydrogenase

Gene mapping 10p15.1 [22] 3p25 [39]

Mass of subunit Unprocessed: 38 556/

mature: 35 436 Da

Monomer: 63 732 Da Cofactors ATP, CoA, Mg2+ O2, Fe2+, ascorbate, 2-oxoglutarate

[18,19]

TPP, Mg2+[26] NAD+[29,30] ATP/GTP, Mg 2+ [21]

K m for CoA-ester 29.5 ± 1.7 lMb[36] 15 lMd[26]

Subcellular localization Peroxisomal

membrane [12–14]?

Peroxisomal matrix [19,20] Peroxisomal matrix [26,27] Peroxisomes

[29,32]? Targeting PTS-2 [22,23] PTS-1 [26]

Stereochemistry Not stereospecifica 3Rfi2S,3R; 3Sfi2R,3S c

[37,38] Not stereospecific [38] Unknowne Heterologous expression

systems

E coli Mammalian cells,

S cerevisiae [26,39]

Mutagenesis studies Yes [22,24,25] No

Structural information Yes [25] TPP binding domain [26,39]

a

As both phytanic acid and phytanoyl-CoA are racemic at position 3, it is supposed that the acyl-CoA synthetase is not stereospecific Whether the activation rates for the R- and S-isomers are different, as shown for the conversion of 2-methyl-branched fatty acids to the corresponding acyl-CoA esters in human liver [40], is not known b K m determined for phytanoyl-CoA with recombinant PAHX, in the presence of equimolar concentrations of SCP-2.cPhytanoyl-CoA hydroxylase is not stereospecific, but the configuration of the methyl-branch at position 3 determines the orientation of the hydroxy-group at position 2 Eventually, only (2R,3S) and (2S,3R) isomers are formed d K m determined for 2-hydroxy-3-methyl-C16-CoA with partially purified enzyme e Although nothing is known about the stereo-specificity of aldehyde dehydrogenases, it can be postulated from all different data concerning the stereochemistry of the a-oxidation pathway that this last step of the reaction sequence is not stereospecific.

diastereoisomers and their ratios are variable and

depend-ent on sample origin As the a-oxidation product of

racemic phytanic acid, pristanic acid, is racemic at

position 2, it seems obvious that both stereoisomers can

undergo a-oxidation without a previous isomerization at

the initial 3-methyl-branch Croes et al [38] provided

indeed evidence that isomerization of the 3-methyl-branch

during a-oxidation does not occur and that the

configur-ation of the methyl-branch is conserved throughout the

whole a-oxidation process It was also demonstrated that

the configuration of the 3-methyl-branch does not

influ-ence the rate of a-oxidation, but determines the

orienta-tion of the 2-hydroxylaorienta-tion This explains the formaorienta-tion

of only the (2S,3R) and (2R,3S) isomers of

2-hydroxy-3-methylhexadecanoyl-CoA by purified peroxisomes, despite

the experimental finding that all four possible isomers

(although each to a different extent) can be metabolized

[38] The data of Croes et al confirm the earlier findings

of Tsai [37], who concluded that the introduction of the

hydroxy group at position 2 is stereospecific and

deter-mined by the configuration of the methyl group at

position 3 The stereochemistry of the a-oxidation

path-way is presented in Fig 3

The lack of stereospecificity of the a-oxidation pathway is

in contrast with the stereospecificity of both the peroxisomal

and mitochondrial b-oxidation systems As a-oxidation of

phytanic acid results in both stereoisomers of pristanic acid,

the produced (2R,6R,10R) isomer has to undergo

racemi-zation at carbon 2 before b-oxidation can take place In

addition, racemization at the other chiral centres is an essential step for the further b-oxidation of the intermediate a-methyl fatty acids [40]

2-HPCL: a thiamine dependent enzyme 2-HPCL identification

After the discovery by Poulos et al [11] of formate as a product of a-oxidation in fibroblasts, a finding which was confirmed in isolated hepatocytes [33], Croes et al found in

1997 that not formate (or CO2) was the primary end product but formyl-CoA [28] This finding led several authors to propose a reaction mechanism in which the other product would be a 2-methyl-branched aldehyde (or pristanal in case phytanic acid is the substrate) Soon, the formation of a 2-methyl-branched aldehyde, using 2-hydroxy-3-methylacyl-CoA or 2-hydroxyphytanoyl-CoA

as precursor, was demonstrated simultaneously by Croes

et al [29] and Verhoeven et al [30]

Foulon et al used 2-hydroxy-3-methylhexadecanoyl-CoA as substrate for studying the third reaction of the a-oxidation pathway, and measured formate (together with formyl-CoA, which is, partly enzymatically, converted to formate) as the reaction product [26]

Subcellular fractionation studies in rat liver demonstra-ted that the lyase activity colocalized with catalase in the peroxisomal fraction [26] Hence, isolation of the pre-sumptive cleavage enzyme was started from the matrix protein fraction of isolated rat liver peroxisomes The purified lyase was made up of four identical subunits of

63 kDa Formyl-CoA and 2-methylpentadecanal (meas-ured by GC-analysis) were identified as reaction products when the enzyme (in the presence of thiamine pyrophos-phate (TPP), see below) was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate Quantitative measurements of both reaction products further confirmed the stoichiometry of the cleavage step Incubations in the presence of NAD+ (a cofactor for fatty aldehyde dehydrogenation [43]) did not alter the amount of formate (formyl-CoA) and 2-methyl-pentadecanal formed, and no conversion of the aldehyde to a fatty acid could be demonstrated indicating that this reaction is performed by

a separate enzyme Hence, as the only activity of the purified enzyme is the specific cleavage of a carbon-carbon bond, it was called 2-hydroxyphytanoyl-CoA lyase or 2-HPCL [26]

An apparent Km of 15 lMfor 2-hydroxy-3-methylhexa-decanoyl-CoA was calculated The pH optimum was between 7.5 and 8.0 [26]

TPP-dependence of 2-HPCL Originally, 2-HPCL had been purified in the absence of TPP and the enzyme lost virtually all of its activity during purification The amino-acid sequences of tryptic peptides from the purified and barely active 2-HPCL suggested that the cleavage enzyme is related to a putative Caenorhabditis elegansprotein that displays homology to bacterial oxalyl-CoA decarboxylases [44,45] These enzymes, which have hitherto only been described in bacteria, catalyse the TPP-dependent decarboxylation of oxalyl-CoA to formyl-CoA

Fig 3 Stereochemistry of the a-oxidation pathway The scheme

rep-resents the a-oxidation pathway of (3R,3S)-methylhexadecanoic acid

and the stereochemical configuration of the intermediates involved.

The numbers indicate the enzymes catalysing the different steps: (1)

acyl-CoA synthetase; (2) phytanoyl-CoA hydroxylase (PAHX); (3)

2-hydroxyphytanoyl-CoA lyase (2-HPCL); (4) aldehyde

dehydro-genase; (5) formyl-CoA hydrolase; (6) acyl-CoA synthetase; and (7)

2-methylacyl-CoA racemase, responsible for the conversion of the

2R-methylacyl-CoA into the 2S-methylacyl-CoA, as only the S-isomer

can undergo b-oxidation.

and CO2 [44,45] This homology suggested that also

2-HPCL might require TPP, an unexpected cofactor for

a-oxidation

In the presence of 0.8 mM Mg2+, optimum activity for

the purified enzyme was reached at 20 lM TPP (Km for

TPP¼ 8.43 lM) Only minor stimulation by TPP was

noted in a fresh liver homogenate (1.3 fold), and a gradually

more potent stimulation of the lyase activity was observed

as the enzyme became more purified Hence, optimal lyase

measurements have to be performed in the presence of TPP

and MgCl2

cDNA and amino-acid sequence

The cDNA sequence of the human lyase contains an open

reading frame of 1734 nucleotides encoding a polypeptide

with a calculated molecular mass of 63 732 Da Similarly

to other TPP-dependent enzymes (e.g bacterial

oxalyl-CoA decarboxylases), a TPP-binding consensus domain

could be identified in the C-terminal part of the lyase The

corresponding peptide sequences of this domain in the

human, mouse and rat enzyme, comply exactly with

the TPP consensus domain of pyruvate decarboxylase of

Saccharomyces cerevisiae, acetolactate synthase of

Escheri-chia coli, oxalyl-CoA decarboxylase of Oxalobacter

formi-genes and the putative oxalyl-CoA decarboxylases of

Caenorhabditis elegans and S cerevisiae [44,45] (Fig 4

[46])

Substrate specificity of 2-HPCL

Recombinant human protein, expressed in mammalian cells

or in a yeast system, clearly exhibited lyase activity, whereas

expression in a bacterial system did not result in a

functionally active enzyme [26]

Study of the substrate specificity of recombinant

human lyase revealed that the enzyme is not only active

towards 2-hydroxy-3-methylhexadecanoyl-CoA (the

analogue of 2-hydroxyphytanoyl-CoA), but also,

although to a minor extent, towards

2-hydroxyoctadeca-noyl-CoA (± 12% of control activity) at equal substrate

concentration The latter compound, however, as well as

2-hydroxyhexadecanoyl-CoA, effected a very strong

inhi-bition on the cleavage of

2-hydroxy-3-methylhexadeca-noyl-CoA, most probably due to competition [39] No

activity at all was seen with

2-hydroxy-3-methylhexadeca-noic acid, 3-methylhexadeca2-hydroxy-3-methylhexadeca-noic acid or 3-methylhexa-decanoyl-CoA, indicating that both a 2-hydroxy group and a CoA-moiety, but not a 3-methyl-branch, are necessary for lyase activity [39]

Identification of novel PTS

At first glance, the Hs 2-HPCL sequence did not contain a C-terminal or N-terminal peroxisome targeting signal (PTS) As the C elegans orthologue ends in a putative PTS1 (SKM) and as PRL, the C-terminal tripeptide of the

S cerevisiae orthologue, had been shown to bind to the human PTS1 import receptor [47], the C-terminal sequence SNM, which is also conserved in the mouse counterpart, was considered to have a targeting function Transfection studies with constructs coding for 2-HPCL fused to GFP revealed that the fluorescence localized to peroxisomes in fibroblasts from PEX5+/– mice and to the cytosol in fibroblasts from PEX5–/–mice [26] The latter mice lack the PTS1 receptor (Pex5p) and do not import PTS1-containing proteins into their peroxisomes [48] As a GFP-construct containing only the last 5 amino acids of 2-HPCL localized

to peroxisomes in fibroblasts from normal mice, we can conclude that targeting information is present within this pentapeptide and that SNM, preceded by a positive charge,

is a hitherto unrecognized PTS1 [26]

Reaction mechanism of 2-HPCL

A 2-hydroxy carboxyl compound (instead of a 2-keto carboxyl compound) is a rather unusual substrate for thiamine dependent decarboxylases In all TPP-dependent reactions described so far, catalysis involves activation of the C2-H of the thiazole ring, followed by a nucleophilic attack at the carbonyl carbon of the substrate [49] By use of nuclear magnetic resonance spectroscopy, it has been shown that in the enzyme-bound state, the C2 proton of TPP is undissociated, but that the protein component dramatically accelerates the deprotonation, producing an intermediate C2 carbanion with a short lifetime [50,51] Most likely, the formation of a carbanion is also required for the cleavage of 2-hydroxy-3-methylacyl-CoAs by 2-HPCL (Fig 5) How-ever, this carbanion will attack carbon 1 of the substrate, which is highly reactive due to the nature of the thioester bond Ultimately this leads to the formation of formyl-CoA and a 2-methyl-branched fatty aldehyde

Fig 4 Alignment of the cofactor-binding consensus domain in TPP-dependent enzymes An alignment [26] is given of the cofactor-binding consensus domain in several TPP-dependent enzymes (Sc PDC: S cerevisiae pyruvate decarboxylase; Ec ALS: E coli acetolactate synthase; Of OCD:

O formigenes oxalyl-CoA decarboxylase) and in Hs 2-HPCL and its homologues in lower organisms (Ce OCD: C elegans putative oxalyl-CoA decarboxylase; Sc OCD: S cerevisiae putative oxalyl-CoA decarboxylase) The TPP-binding consensus motif, here represented with 10 residues upstream and downstream, is defined as G-D-G-x-(24–27)-N-N [46] About 10 residues downstream of the G-D-G sequence, a negatively charged amino acid is present (E or D), followed about 5 and 11 residues further by a generally conserved alanine and proline residue, respectively Immediately preceding the N-N sequence is a cluster of 6 or 7 largely hydrophobic side-chains.

As 2-hydroxy-acyl-CoA esters seem to be unusual

substrates for TPP-dependent enzymes, Jones et al [52]

proposed another mechanism for a-oxidation from the

conversion of 2-hydroxyphytanoyl-CoA onwards This

would involve hydrolysis of the CoA-ester (peroxisomal

thioesterases have been described [53]) and a subsequent

oxidation generating 2-ketophytanic acid, which would

then be cleaved by 2-HPCL, the enzyme described by us

[26] This hypothesis would turn 2-HPCL into a not so

unusual TPP-dependent enzyme as its substrate would

then be a 2-keto-compound However, the activity of

the required thioesterases toward the proposed substrate

has never been demonstrated and the 2-hydroxyacid

oxidase, present in kidney, is only active on

L-2-hydroxyphytanic acid [54], whereas the activity of

2-HPCL vs 2-hydroxy-3-methylacyl-CoA has

unequivo-cally been proven Moreover, if, according to the

hypothesis of Jones et al [52], a thioesterase and a

2-hydroxyacid oxidase would be involved, no

formyl-CoA/formate would be produced This would be in

contrast with the solid findings of several authors

[11,18,19,28,33,55]

Mapping of the 2-HPCL gene The human 2-HPCL gene has been mapped to chromo-some 3p25 (Foulon V., Vermeesch J., Mannaerts G.P., Casteels M., Van Veldhoven P.P.; unpublished results) The complete Hs 2-HPCL gene spans 40.8 kb and contains

17 exons, with intron sizes ranging from 190 bp to 4700 bp All exon-intron boundaries are conform to the consensus rules [56], ending in an AG doublet and starting with a GT pair

Gene defects of 2-HPCL associated with ARD?

Although several diseases are known to be associated with 3p25, none of these appear to be linked to 2-HPCL Moreover, up till now no patients with a deficient 2-HPCL, which would probably result in a clinical picture similar to ARD, have been identified The mapping of the 2-HPCL gene is a first step towards the finding and diagnosis of such patients

Deficient breakdown of phytanic acid Elevated serum levels of phytanic acid are typical for patients with an isolated defective a-oxidation but can also

be seen in patients with peroxisome biogenesis disorders

In the latter patients the accumulation of phytanic acid is only one of the features present [4]

The most typical clinical picture of an isolated defect in phytanic acid breakdown is described as ARD [2,3] The gradual accumulation of phytanic acid in serum and tissues

of these patients results only in the second or third decade in distinct symptoms Virtually all patients show retinitis pigmentosa, night blindness and anosmia (deficient smelling sensation; 80% of ARD patients) In addition, polyneuro-pathy (60%), deafness (60%), ataxia (50%) and ichtyosis (20%) are quite common (for a review, see Wierzbicki et al [3]) A prerequisite for the diagnosis of ARD is the presence

of an elevated serum level of phytanic acid (above 200 lM

whereas normal phytanic acid levels in serum are below

30 lM) However, there seems to be no strict correlation between the level of phytanic acid accumulation and the severity of the clinical symptoms Interestingly, 30–40%

of the patients are born with an absence of one of the metacarpals or metatarsals (bone in the hand or forefoot, respectively)

The pathophysiology and the cause of the retinal and specific neurological manifestations of ARD remain at present unknown Feeding control animals excessive amounts of phytol can lead to similar severe neurological symptoms as in ARD, indicating that at least some of the symptoms in ARD might be directly related to an accumu-lation of phytanic acid Most obviously, the study of animal models for ARD will help to clarify the pathogenetic mechanisms of this disease

The clinical spectrum of ARD can be ascribed to different molecular and genetic defects [57] Probably most frequent

is a defect at the level of PAHX, mapped to chromosome 10p15.1 [22,23] However, some patients show a low or absent phytanoyl-CoA hydroxylase activity, but no muta-tion in PAHX Van den Brink et al [58] described in 2 such

Fig 5 Generation of a carbanion in enzyme-bound TPP and proposed

reaction mechanism for 2-HPCL In order to react with the substrate,

the C2-H of TPP must be activated by the protein component A key

function for this activation is the interaction of a conserved glutamate

[50,51] with the N1¢ atom of the coenzyme, resulting in an increased

basicity of its 4¢ amino group, facilitating the deprotonation of the C2.

patients, who had been clinically diagnosed as ARD, a

mutation in the gene encoding PEX7p, the PTS2 import

receptor, apparently resulting in a deficient peroxisomal

import of the PTS2 containing PAHX These patients had

normal peroxisomes, normal peroxisomal b-oxidation, no

or very low PAHX activity, and deficient plasmalogen

synthesis, which is also dependent on an intact import of

PTS2 containing proteins So far, PEX7 mutations were

known to cause rhizomelic chondrodysplasia punctata

(RCDP), resulting in a short lifespan [59–61], but they can

apparently also result in a much milder phenotype with late

onset Additionally, Ferdinandusse et al described two

atypical ÔARDÕ patients, who eventually appeared to have a

racemase deficiency [62] (see legend to Fig 3) Nevertheless,

in some patients with the clinical syndrome of ARD none of

these specific molecular defects could be found and the

genetic basis of the disease in these patients awaits to be

defined

Conclusions and perspectives

2-HPCL is the first mammalian peroxisomal enzyme that is

TPP dependent This finding raises several questions

discussed below

(a) The TPP dependence of 2-HPCL renders the

a-oxidation pathway thiamine dependent as a whole This

could imply that the thiamine status of the cell would

influence the a-oxidation process, but so far no indication

pointing to this hypothesis can be found in the literature

Preliminary experiments with cultured C6-glia cells or

control human fibroblasts in thiamine-deficient conditions

(generated either by the addition of oxythiamine to the

growth medium, or by culturing cells in thiamine-depleted

medium) showed a decrease of the overall fluxthrough

the a-oxidation pathway (V Foulon, M Casteels &

P.P Van Veldhoven, unpublished results) Whether

overall a-oxidation would be deficient in patients with

thiamine deficiency as, e.g thiamine responsive

megalo-blastic anemia (TRMA), and whether this would lead to an

accumulation of phytanic acid in these patients, remains to

be investigated

(b) A TPP dependent reaction in peroxisomes requires

the presence of thiamine or thiamine pyrophosphate

inside the peroxisome It was shown recently that the

thiamine transporter SLC19A2, which is deficient in

TRMA, is not only present on the plasma membrane

but also on the mitochondrial membrane [63] No report

was made however, on the presence of this transporter

(or one of his homologues) on the peroxisomal

mem-brane As 2-HPCL is the first mammalian peroxisomal

enzyme described to be TPP-dependent, the mechanism

for the import of thiamine/TPP into peroxisomes remains

to be explored

Acknowledgements

This work was supported by grants from the ÔGeconcerteerde

onderzoeksacties van de Vlaamse GemeenschapÕ (GOA 94/98–12 and

GOA 99/03–09) and from the ÔFonds voor Wetenschappelijk

Onder-zoek-VlaanderenÕ (G-0239.98, G.0164.96 N and G.0115.02) V.F was

supported by a fellowship from the ÔFonds voor Wetenschappelijk

Onderzoek-VlaanderenÕ.

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