Stanny Asselberghs

Peroxisomal β-oxidation of 2-methyl-branched acyl-CoA esters stereospecific recognition of the 2S-methyl compounds by trihydroxycoprostanoyl-CoA oxidase and pristanoyl-CoA oxidase

Trihydroxycoprostanoyl‐CoA oxidase and pristanoyl‐CoA oxidase, purified from rat liver, both catalyse the desaturation of 2‐methyl‐branched acyl‐CoAs. Upon incubation with the pure isomers of 2‐methylpentadecanoyl‐CoA, both enzymes acted only on the S‐isomer. The R‐isomer inhibited trihydroxycoprostanoyl‐CoA oxidase but did not affect pristanoyl‐CoA oxidase. The activity of both enzymes was suppressed by 3‐methylheptadecanoyl‐CoA. Valproyl‐CoA and 2‐ethylhexanoyl‐CoA, however, did not influence the oxidases. Although only one isomer of 25R,S‐trihydroxycoprostanoyl‐CoA was desaturated by trihydroxycoprostanoyl‐CoA oxidase, isolated peroxisomes were able to act on both isomers, suggesting the presence of a racemase in these organelles. Given the opposite stereoselectivity of the 26‐cholesterol hydroxylase and of the oxidase, the racemase is essential for bile acid formation.

Breakdown of 2-hydroxylated straight chain fatty acids via peroxisomal 2-hydroxyphytanoyl-CoA lyase: a revised pathway for the alpha-oxidation of straight chain fatty acids.

2-Hydroxyfatty acids, constituents of brain cerebrosides and sulfatides, were previously reported to be degraded by an α-oxidation system, generating fatty acids shortened by one carbon atom. In the current study we used labeled and unlabeled 2-hydroxyoctadecanoic acid to reinvestigate the degradation of this class of lipids. Both in intact and broken cell systems formate was identified as a main reaction product. Furthermore, the generation of an n–1 aldehyde was demonstrated. In permeabilized rat hepatocytes and liver homogenates, studies on cofactor requirements revealed a dependence on ATP, CoA, Mg2+, thiamine pyrophosphate, and NAD+. Together with subcellular fractionation data and studies on recombinant enzymes, this led to the following picture. In a first step, the 2-hydroxyfatty acid is activated to an acyl-CoA; subsequently, the 2-hydroxy fatty acyl-CoA is cleaved by 2-hydroxyphytanoyl-CoA lyase, to formyl-CoA and an n–1 aldehyde. The severe inhibition of formate generation by oxythiamin treatment of intact fibroblasts indicates that cleavage through the thiamine pyrophosphate-dependent 2-hydroxyphytanoyl-CoA lyase is the main pathway for the degradation of 2-hydroxyfatty acids. The latter protein was initially characterized as an essential enzyme in the peroxisomal α-oxidation of 3-methyl-branched fatty acids such as phytanic acid. Our findings point to a new role for peroxisomes in mammals, i.e. the breakdown of 2-hydroxyfatty acids, at least the long chain 2-hydroxyfatty acids. Most likely, the more abundant very long chain 2-hydroxyfatty acids are degraded in a similar manner.

Formation of a 2-methyl-branched fatty aldehyde during peroxisomal α-oxidation

In the final reaction of peroxisomal α‐oxidation of 3‐methyl‐branched fatty acids a 2‐hydroxy‐3‐methylacyl‐CoA intermediate is cleaved to formyl‐CoA and a hitherto unidentified product. The release of formyl‐CoA suggests that the unidentified product may be a fatty aldehyde. When purified rat liver peroxisomes were incubated with 2‐hydroxy‐3‐methylhexadecanoyl‐CoA 2‐methylpentadecanal was indeed formed. The production rates of formyl‐CoA (measured as formate) and of the aldehyde were in the same range. While the production of formate remained unaltered in the presence of NAD+, the amount of 2‐methylpentadecanal was decreased, which was accompanied by the formation of 2‐methylpentadecanoic acid. These data indicate that (1) during α‐oxidation the 2‐hydroxy‐3‐methylacyl‐CoA is cleaved to a 2‐methyl‐branched aldehyde and formyl‐CoA and (2) liver peroxisomes are capable of converting this aldehyde to a 2‐methyl‐branched fatty acid.

Further studies on the substrate spectrum of phytanoyl-CoA hydroxylase implications for Refsum disease?

Refsum disease is a peroxisomal disorder characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy, ataxia, and an accumulation of phytanic acid in plasma and tissues. Approximately 45% of cases are caused by mutations in phytanoyl-CoA hydroxylase (PAHX), the enzyme catalyzing the second step in the peroxisomal α-oxidation of 3-methyl-branched fatty acids. To study the substrate specificity of human PAHX, different 3-alkyl-branched substrates were synthesized and incubated with a recombinant polyhistidine-tagged protein. The enzyme showed activity not only toward racemic phytanoyl-CoA and the isomers of 3-methylhexadecanoyl-CoA, but also toward a variety of other mono-branched 3-methylacyl-CoA esters with a chain length down to seven carbon atoms. Furthermore, PAHX hydroxylated a 3-ethylacyl-CoA quite well, whereas a 3-propylacyl-CoA was a poor substrate. Hydroxylation of neither 2- or 4-methyl-branched acyl-CoA esters, nor long or very long straight-chain acyl-CoA esters could be detected. The results presented in this paper show that the substrate specificity of PAHX, with regard to the length of both the acyl-chain and the branch at position 3, is broader than expected. Hence, Refsum disease might be characterized by an accumulation of not only phytanic acid but also other 3-alkyl-branched fatty acids.

Activation of 3-methyl-branched fatty acids in rat liver.

1. Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes. 2. Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes. 3. Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data. On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2-methylpalmitic and palmitic acid. The latter three however, tended to coelute together, although often not in an identical manner.

1-O-Hexadecyl-2-desoxy-2-amino-sn-glycerol, a substrate for human sphingosine kinase.

The substrate specificity of human sphingosine kinase was investigated using a bacterially expressed poly(His)-tagged protein. Only the D-erythro isomer of the sphingoid bases, sphinganine and sphingenine, was effectively phosphorylated. Long chain 1-alkanols, alkane-1,2-diols, 2-amino-1-alkanol or 1-amino-2-alkanol and short chain 2-amino-1,3-alkanediols were very poor substrates, indicating that the kinase is recognizing the chain length and the position of the amino and secondary hydroxy group. A free hydroxy group at carbon 3 is not a prerequisite, however, since 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol was an efficient substrate with an apparent K(m) value of 3.8 microM (versus 15.7 microM for sphingenine). This finding opens new perspectives to design sphingosine kinase inhibitors. It also calls for some caution since it cannot be excluded that this ether lipid analogue is formed from precursors that are frequently used in research on platelet activating factor or from phospholipid analogues which are less prone to degradation.

Comparison of Fatty-acid Alpha-oxidation By Rat Hepatocytes and By Liver-microsomes Fortified With Nadph, Fe3+ and Phosphate

Rat liver microsomes, when fortified with NADPH, Fe3+ and phosphate, can catalyze the oxidative decarboxylation (α-oxidation) of 3-methyl-substituted fatty acids (phytanic and 3-methylheptadecanoic acids) at rates that equal 60–70% of those observed in isolated hepatocytes (Huang, S., Van Veldhoven, P.P., Vanhoutte, F., Parmentier, G., Eyssen, H.J., and Mannaerts, G.P., 1992,Arch. Biochem. Biophys. 296, 214–223). In the present study we set out to identify and compare the products and possible intermediates of α-oxidation formed in rat hepatocytes and by rat liver microsomes. In the presence of NADPH, Fe3+ and phosphate, microsomes decarboxylated not only 3-methyl fatty acids but also 2-methyl fatty acids and even straight chain fatty acids. The decarboxylation products of 3-methylheptadecanoic and palmitic acids were purified by highperformance liquid chromatography and identified by gas chromatography/mass spectrometry as 2-methyl-hexadecanoic and pentadecanoic acids, respectively. Inclusion in the incubation mixtures of glutathione plus glutathione peroxidase inhibited decarboxylation by more than 90%, suggesting that a 2-hydroperoxy fatty acid is formed as a possible intermediate. However, we have not yet been able to unequivocally identify this intermediate. Instead, several possible rearrangement metabolites were identified. In isolated rat hepatocytes incubated with 3-methylheptadecanoic acid, the formation of the decarboxylation product, 2-methylhexadecanoic acid, was demonstrated, but no accumulation of putative intermediates or rearrangement products was observed. Our data do not allow us to draw conclusions on whether the reconstituted microsomal system is representative of the cellular α-oxidation system. However, the results we obtained with [3-3H]-labelled fatty acids indicate that during α-oxidation in intact cells the hydrogen at carbon-3, which carries the methyl branch, is not attacked.

Phytol-induced pathology in 2-hydroxyacyl-CoA lyase (HACL1) deficient mice. Evidence for a second non-HACL1-related lyase

2-Hydroxyacyl-CoA lyase (HACL1) is a key enzyme of the peroxisomal α-oxidation of phytanic acid. To better understand its role in health and disease, a mouse model lacking HACL1 was investigated. Under normal conditions, these mice did not display a particular phenotype. However, upon dietary administration of phytol, phytanic acid accumulated in tissues, mainly in liver and serum of KO mice. As a consequence of phytanic acid (or a metabolite) toxicity, KO mice displayed a significant weight loss, absence of abdominal white adipose tissue, enlarged and mottled liver and reduced hepatic glycogen and triglycerides. In addition, hepatic PPARα was activated. The central nervous system of the phytol-treated mice was apparently not affected. In addition, 2OH-FA did not accumulate in the central nervous system of HACL1 deficient mice, likely due to the presence in the endoplasmic reticulum of an alternate HACL1-unrelated lyase. The latter may serve as a backup system in certain tissues and account for the formation of pristanic acid in the phytol-fed KO mice. As the degradation of pristanic acid is also impaired, both phytanoyl- and pristanoyl-CoA levels are increased in liver, and the ω-oxidized metabolites are excreted in urine. In conclusion, HACL1 deficiency is not associated with a severe phenotype, but in combination with phytanic acid intake, the normal situation in man, it might present with phytanic acid elevation and resemble a Refsum like disorder.

Stabilisation and partial purification of Triton X‐100 solubilised trihydroxycoprostanoyl‐coa synthetase from rat liver

The stability of rat hepatic trihydroxycoprostanoyl‐CoA syntethase was studied in its native membrane environment and after solubilisation by Triton X‐100, and compared to that of choloyl‐CoA synthetase. The lability of both delipidated enzymes could be suppressed by high concentrations of polyols such as sucrose and glucose. Addition of phospholipids to the assay mixtures was necessary to restore the activity of the stabilized enzymes. For further chromatographic separations, the addition of the hydrotrope Triton H‐66 to the glucose‐stabilized Triton X‐100 solubilised synthetases improved their recovery on different matrices. Gel filtration revealed a native molecular mass of the Triton X‐100/Triton H‐66/protein micelles of 212 and 207 kDa for choloyl‐CoA synthetase and trihydroxycoprostanoyl‐CoA synthetase respectively.