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

REVIEW ARTICLE 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-andb-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) Eur. J. Biochem. 270, 1619–1627 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03534.x 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 CO 2 ,as was generally believed, but also formate [11]. Up till then only CO 2 had 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 CO 2 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-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 O 2 , iron, 2-oxoglutarate, ascorbate, ATP/GTP and Mg 2+ [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. 1620 M. Casteels et al. (Eur. J. Biochem. 270) Ó FEBS 2003 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 matrix according to Croes et al.[29]andinthe 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 of 14 C-formate to CO 2 (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 of 14 C-formate to 14 CO 2 in 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 Accession number O14832 Q9UJ83 Gene mapping 10p15.1 [22] 3p25 [39] Mass of subunit Unprocessed: 38 556/ mature: 35 436 Da Monomer: 63 732 Da Cofactors ATP, CoA, Mg 2+ O 2 ,Fe 2+ , ascorbate, 2-oxoglutarate [18,19] TPP, Mg 2+ [26] NAD + [29,30] ATP/GTP, Mg 2+ [21] K m for CoA-ester 29.5 ± 1.7 lM b [36] 15 lM d [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 stereospecific a 3Rfi2S,3R;3Sfi2R,3S c [37,38] Not stereospecific [38] Unknown e 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. c Phytanoyl-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. Ó FEBS 2003 Alpha-oxidation and its TPP dependence (Eur. J. Biochem. 270) 1621 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-hydroxylation. This explains the formation 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 CO 2 ) 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 l M for 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 elegans protein 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. 1622 M. Casteels et al. (Eur. J. Biochem. 270) Ó FEBS 2003 and CO 2 [44,45]. This homology suggested that also 2-HPCL might require TPP, an unexpected cofactor for a-oxidation. In the presence of 0.8 m M Mg 2+ , optimum activity for the purified enzyme was reached at 20 l M TPP (K m for TPP ¼ 8.43 l M ). 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 MgCl 2 . 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-methylhexadecanoic 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 +/– miceandtothecytosolin 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. Ó FEBS 2003 Alpha-oxidation and its TPP dependence (Eur. J. Biochem. 270) 1623 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 l M whereas normal phytanic acid levels in serum are below 30 l M ). 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 isadefectatthelevelofPAHX, 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. 1624 M. Casteels et al. (Eur. J. Biochem. 270) Ó FEBS 2003 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 flux through 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. 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(2002) Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate. BMC Biochem. 3,8. Ó FEBS 2003 Alpha-oxidation and its TPP dependence (Eur. J. Biochem. 270) 1627 . REVIEW ARTICLE Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence Minne Casteels, Veerle Foulon, Guy P. Mannaerts and Paul P a-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO 2 , cofactor requirements, subcellular localization and formation