Kathleen Croes

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.

Production of formyl-CoA during peroxisomal α-oxidation of 3-methyl-branched fatty acids

α‐Oxidation of 3‐methyl‐substituted fatty acids was studied in purified rat liver peroxisomes. The experiments revealed that formyl‐CoA is formed during the α‐oxidation process. The amount of formyl‐CoA found constituted 2–5% of the amount of formate formed. Under the conditions used, no activation of exogenously added formate occurred in purified peroxisomes, whereas 95.5% of added synthetic formyl‐CoA was converted to formate. These data indicate that during α‐oxidation first formyl‐CoA is formed, which is then hydrolysed to formate.

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.

Aminotriazole is a potent inhibitor of α-oxidation of 3-methyl-substituted fatty acids in rat liver

The production of CO2 and formate in isolated rat hepatocytes incubated in the presence of 3-methyl[1-14C]margaric acid was investigated. Production rates of formate were approximately 4-fold lower than those of CO2. Aminotriazole (3-amino-1, 2, 4-triazole), an irreversible inhibitor of catalase, potently suppressed alpha-oxidation of 3-methylmargaric acid, whereas beta-oxidation of palmitate, 2-methylpalmitate and trihydroxycoprostanic acid and conversion of exogenously added formate to CO2 were not or only slightly affected. This shows that aminotriazole is not only an inhibitor of catalase, but also of alpha-oxidation of 3-methyl-substituted fatty acids.

2-Methylacyl racemase: a coupled assay based on the use of pristanoyl-CoA oxidase/peroxidase and reinvestigation of its subcellular distribution in rat and human liver

Because of the 2S-methyl-stereospecificity of the acyl-CoA oxidases acting on the CoA esters of 2-methyl-branched fatty carboxylates such as pristanic acid and the side chain of trihydroxycoprostanic acid (Van Veldhoven P.P., Croes K., Asselberghs S., Herdewijn P. and Mannaerts G.P. (1996) FEBS Lett. 388, 80-84), naturally occurring 2R-pristanic acid and 25R- (corresponding to 2R in the side chain) trihydroxycoprostanic acid, after activation to their CoA-esters, need to be racemized to the S-isomers before they can be degraded by peroxisomal beta-oxidation. A coupled assay to measure 2-methyl-acyl racemases was developed by using purified rat pristanoyl-CoA oxidase. Upon incubation of rat and human liver homogenates with 2R-methyl-pentadecanoyl-CoA, the formed 2S-methyl isomer was desaturated by an excess of added oxidase and the concomitant production of hydrogen peroxide was monitored by means of peroxidase in the presence of a suitable hydrogen donor. Application of this assay to subcellular fractions of rat liver revealed the presence of racemase activity not only in mitochondria, as described by Schmitz W., Albers C., Fingerhut R. and Conzelmann E. (Eur. J. Biochem. (1995) 231, 815-822), but also in peroxisomes and cytosol. A similar distribution was seen in human liver. In rat the highest activities were found in liver, followed by Harderian gland, kidney and intestinal mucosa.

Peroxisomal localization of α-oxidation in human liver

It was found that α-oxidation in rat liver is a peroxisomal process, consisting of an activation, a 2-hydroxylation, and a reaction leading to the production of formate. α-Oxidation of 3-methyl-substituted fatty acids was quantified by measuring the production of formate and CO2, and the production of a 2-hydroxy-3-methylacyl-CoA-intermediate was demonstrated. We wanted to extend these findings to human liver, in view of the controversy over the subcellular localization of α-oxidation in man.In homogenates from human liver, rates of α-oxidation were highest when measured in the presence of ATP, CoA, Mg2+, 2-oxoglutarate, ascorbate and Fe2+. In subcellular fractions prepared by differential centrifugation and in fractions obtained after subfractionation of a peroxisome-enriched fraction on a Percoll gradient, production of formate and of a 2-hydroxy-3-methylacyl-CoA intermediate coincided with the peroxisomal marker catalase. In broken fractions, production of CO2 was almost negligible as compared to formate production.We conclude from our findings that in human liver, as in rat liver, α-oxidation of 3-methyl-substituted fatty acids is a peroxisomal process, consisting of an activation reaction, a 2-hydroxylation reaction and a reaction or reactions leading to the generation of formate as a primary product which is subsequently converted to CO2. Furthermore activation, 2-hydroxylation and generation of formate appear to be coupled (see Casteels et al 1996). These data demonstrate that Refsum disease should indeed be classified as a peroxisomal disease.

Hepatic α-Oxidation of Phytanic Acid

Synthetic 3-methyl-branched chain fatty acids were used to decipher the breakdown of phytanic acid. Based on results obtained in intact or permeabilized rat hepatocytes, rat liver homogenates or subcellular fractions, a revised α-oxidation pathway is proposed which appears to be functioning in man as well. In a first step, the 3-methyl-branched chain fatty acid is activated by an acyl-CoA synthetase. This reaction requires CoA, ATP and Mg2+. Subsequently, the acyl-CoA ester is hydroxylated at position 2 by a peroxisomal dioxygenase. This step is dependent on α-oxoglutarate, ascorbate (or glutathione), Fe2+ and O2. The 2-hydroxy-3-methylacyl-CoA intermediate is cleaved by a peroxisomal lyase to formyl-CoA and a 2-methyl-branchedfatty aldehyde. Formyl-CoA is (partly enzymically) hydrolyzed to formate, which is then converted, most likely in the cytosol, to CO2. In the presence of NAD+, the aldehyde is dehydrogenated to a 2-methyl-branched fatty acid, presumably by a peroxisomal aldehyde dehydrogenase. This acid can—after activation—be degraded via a D-specific peroxisomal β-oxidation system.

ALPHA -OXIDATION OF 3-METHYL-BRANCHED FATTY ACIDS : A REVISED PATHWAY CONFINED TO PEROXISOMES

c~-Oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), a 3-methyl-branched isoprenoid-derived fatty acid, was generally believed to consist of a hydroxylation in the 2-position, followed by an oxidative decarboxylation generating CO 2 and a fatty acid shortened by one carbon. Until recently, however, the (z-oxidation process has remained a matter of debate as to its subcellular localization, the reaction intermediates, and the cofactors involved (1). The observation that formate is formed during o~-oxidation of phytanic acid in human skin fibroblasts (2) has been the basis for the recent unraveling of the cz-oxidation process. For our study, we used synthetic 3-methyl-branched fatty acids that were shown to be valid substitutes for phytanic acid in the study of the (z-oxidation process. As in human fibroblasts, formate was formed during a-oxidation of 3-methyl-branched fatty acids in intact rat hepatocytes (3) and was shown to be the first product which is then converted to CO 2, presumably in an NAD+-dependent cytosolic process (4). Consequently, o~-oxidation was assessed by the sum of formate and CO 2 in all further experiments. When studied in permeabilized rat hepatocytes and rat liver homogenates, the a-oxidation process required ATE CoA, Mg 2+, Fe 2+, ascorbate, and 2-oxoglutarate (4). Subsequent experiments in subcellular fractions of rat liver demonstrated that this set of cofactors reflects two steps: first, activation by an acyl-CoA synthetase requiring ATR CoA, and Mg 2÷ and then 2-hydroxylation of the resulting 3-methylacylCoA by a 3-methylacyl-CoA hydroxylase requiring Fe 2÷, ascorbate, and 2-oxoglutarate (4). The whole 0~-oxidation pathway up to the formation of formate was clearly a peroxisomal process (4). Mihalik et al. (5) came to the same conclusion using a different approach. Furthermore, our results suggest that the 2-hydroxylation is a membrane-bound process (4). The 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde by a 2hydroxy-3-methylacyl-CoA lyase, probably located in the peroxisomal matrix and requiring no additional cofactors. In peroxisomes formyl-CoA is actively converted to formate (6), Lipids 34, S159 (1999). *To whom correspondence should be addressed at Katholieke Universiteit Leuven, Campus Gasthuisberg, Afdeling Farmakologie, Herestraat 49, B3000 Leuven, Belgium. while the 2-methyl-branched fatty aldehyde is converted to a 2-methyl-branched fatty acid in the presence of NAD + (7). After activation to its CoA-ester, the 2-methyl-branched fatty acid can be degraded by peroxisomal ~-oxidation. Our studies show that in rat liver the whole c~-oxidation process up to the formation of formate and a 2-methylbranched fatty acid consists of five enzymatic steps, which are all confined to peroxisomes.