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• W2138889171 abstract "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. 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. Human phytanoyl-CoA hydroxylase (PAHX) is an iron- and 2-oxoglutarate-dependent dioxygenase that catalyzes a key step in the metabolic degradation of 3-methyl-branched fatty acids such as phytanic acid. Due to the presence of a 3-methyl group, these substrates cannot be degraded by the normal β-oxidation pathway. Instead, they are shortened by one carbon atom in a four-step α-oxidation process, yielding 2-methyl-branched fatty acids, which are substrates for subsequent β-oxidation cycles (1Mannaerts G.P. Van Veldhoven P.P. Casteels M. Peroxisomal lipid degradation via α- and β-oxidation in mammals.Cell Biochem. Biophys. 2000; 32: 73-87Crossref PubMed Scopus (100) Google Scholar).In the last decade, the different intermediates and cofactors of the α-oxidation pathway have been characterized (2Poulos A. Sharp P. Singh H. Johnson D.W. Carey W.F. Easton C. Formic acid is a product of the α-oxidation of fatty acids by human skin fibroblasts: deficiency of formic acid production in peroxisome-deficient fibroblasts.Biochem. J. 1993; 292: 457-461Crossref PubMed Scopus (55) Google Scholar, 3Croes K. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Evidence for the importance of iron in the α-oxidation of 3-methyl-substituted fatty acids in the intact cell.Biochim. Biophys. Acta. 1995; 1255: 63-67Crossref PubMed Scopus (10) Google Scholar, 4Mihalik S.J. Rainville A.M. Watkins P.A. Phytanic acid α-oxidation in rat liver peroxisomes. Production of α-hydroxyphytanoyl-CoA and formate is enhanced by dioxygenase cofactors.Eur. J. Biochem. 1995; 232: 545-551Crossref PubMed Scopus (100) Google Scholar, 5Croes K. Casteels M. Mannaerts G.P. Van Veldhoven P.P. α-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3-methyl-CoA intermediate.Eur. J. Biochem. 1996; 240: 674-683Crossref PubMed Scopus (64) Google Scholar, 6Croes K. Van Veldhoven P.P. Mannaerts G.P. Casteels M. Production of formyl-CoA during peroxisomal α-oxidation of 3-methyl-branched fatty acids.FEBS Lett. 1997; 407: 197-200Crossref PubMed Scopus (44) Google Scholar, 7Croes K. Casteels M. Asselberghs S. Herdewijn P. Mannaerts G.P. Van Veldhoven P.P. Formation of a 2-methyl-branched fatty aldehyde during peroxisomal α-oxidation.FEBS Lett. 1997; 412: 643-645Crossref PubMed Scopus (43) Google Scholar, 8Verhoeven N.M. Schor D.S.M. ten Brink H.J. Wanders R.J.A. Jakobs C. Resolution of the phytanic acid α-oxidation pathway: identification of pristanal as product of the decarboxylation of 2-hydroxyphytanoyl-CoA.Biochem. Biophys. Res. Commun. 1997; 237: 33-36Crossref PubMed Scopus (48) Google Scholar, 9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar). Evidence has been found that α-oxidation is localized to peroxisomes, and several key enzymes [PAHX (10Jansen G.A. Ofman R. Ferdinandusse S. Ijlst L. Muijsers A. Skjeldal O.A. Stokke O. Jakobs C. Besley G.T. Wraith J.E. Wanders R.J.A. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.Nat. Genet. 1997; 17: 190-193Crossref PubMed Scopus (240) Google Scholar, 11Mihalik S.J. Morrell J.C. Kim D. Sacksteder K.A. Watkins P.A. Gould S.J. Identification of PAHX, a Refsum disease gene.Nat. Genet. 1997; 17: 185-189Crossref PubMed Scopus (196) Google Scholar), and 2-hydroxyphytanoyl-CoA lyase/2-HPCL (12Foulon V. Antonenkov V.D. Croes K. Waelkens E. Mannaerts G.P. Van Veldhoven P.P. Casteels M. Purification and molecular cloning of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal enzyme that catalyzes the carbon-carbon bond cleavage during α-oxidation of 3-methyl-branched fatty acids.Proc. Natl. Acad. Sci. USA. 1999; 96: 10039-10044Crossref PubMed Scopus (94) Google Scholar)] have been identified and cloned. Phytanic acid is at present the only known physiological substrate of (hepatic) α-oxidation in humans. It is a conversion product of phytol, the side chain of chlorophyll, and is taken up with the diet in ruminant fat and dairy products. Other potential substrates for α-oxidation are retinoic acid, dolichoic acid, and the terminally oxidized isoprenoid moieties of prenylated proteins, although data supporting this contention are still missing.In humans, the plasma level of phytanic acid is normally low (<30 μM). Accumulation is typically seen in adult Refsum disease (ARD) and, to a lesser extent, in generalized peroxisome biogenesis disorders. ARD is an autosomal recessive syndrome, clinically characterized by retinitis pigmentosa, peripheral neuropathy, and cerebellar ataxia, usually presenting in the second or third decade of life. Eliminating phytanic acid from the diet usually results in an arrest of the progress of the disease and even in a regression of the peripheral neuropathy, indicating that at least some of the symptoms observed in ARD are directly correlated with the accumulation of this 3-methyl-branched fatty acid in plasma and tissues (13Wanders R.J.A. Jakobs C. Skjeldal O.H. Refsum disease.in: Scriver C.R. Beaudet A.L. Valle D. Sly W.S. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 3303-3322Google Scholar). Older literature refers also to the accumulation of mono- and tri-unsaturated analogs of phytanic acid in sera and urine of patients with ARD (14Dulaney J.T. Williams M. Evans J.E. Costello C.E. Kolodny E.H. Occurrence of novel branched-chain fatty acids in Refsum's disease.Biochim. Biophys. Acta. 1978; 529: 1-12Crossref PubMed Scopus (13) Google Scholar). These analogs, which are incorporated in phospholipids and neutral lipids, might well be geranylgeraniol-derived products.In 45% of patients affected with ARD, mutations have been found in the cDNA encoding PAHX, and deficiencies in hydroxylase activity resulting from these mutations have been demonstrated (10Jansen G.A. Ofman R. Ferdinandusse S. Ijlst L. Muijsers A. Skjeldal O.A. Stokke O. Jakobs C. Besley G.T. Wraith J.E. Wanders R.J.A. Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.Nat. Genet. 1997; 17: 190-193Crossref PubMed Scopus (240) Google Scholar, 11Mihalik S.J. Morrell J.C. Kim D. Sacksteder K.A. Watkins P.A. Gould S.J. Identification of PAHX, a Refsum disease gene.Nat. Genet. 1997; 17: 185-189Crossref PubMed Scopus (196) Google Scholar, 15Jansen G.A. Hogenhout E.M. Ferdinandusse S. Waterham H.R. Ofman R. Jakobs C. Skjeldal O.H. Wanders R.J.A. Human phytanoyl-CoA hydroxylase: resolution of the gene structure and the molecular basis in Refsum's disease.Hum. Mol. Genet. 2000; 9: 1195-1200Crossref PubMed Scopus (63) Google Scholar). Human PAHX, encoded by a gene on chromosome 10p15.1 (11Mihalik S.J. Morrell J.C. Kim D. Sacksteder K.A. Watkins P.A. Gould S.J. Identification of PAHX, a Refsum disease gene.Nat. Genet. 1997; 17: 185-189Crossref PubMed Scopus (196) Google Scholar), is a 38.5 kDa peroxisomal matrix protein, containing a cleavable peroxisome-targeting sequence, type 2 (PTS2). In the presence of Fe2+, 2-oxoglutarate, ascorbate, Mg2+, and ATP or GTP, the enzyme catalyzes the conversion of 3-methylacyl-CoA esters to the corresponding 2-hydroxy-3-methylacyl-CoA intermediates. Based on these cofactor and cosubstrate requirements, as well as on the comparison of the amino acid sequence and the predicted three-dimensional structure, PAHX was classified as a 2-oxoglutarate/ascorbate-dependent non-heme ferrous ion dioxygenase. As such, PAHX belongs to a class of enzymes that includes propyl hydroxylase, lysyl hydroxylase, aspartyl β-hydroxylase, and γ-butyrobetaine hydroxylase (16De Carolis E. De Luca V. 2-Oxoglutarate-dependent dioxygenase and related enzymes: biochemical characterization.Phytochemistry. 1994; 36: 1093-1107Crossref PubMed Scopus (93) Google Scholar). The O2-dependent reactions catalyzed by these enzymes are characterized by the fact that one atom of molecular oxygen is incorporated into the substrate, while the other atom of oxygen is incorporated in the cosubstrate 2-oxoglutarate, resulting in the subsequent formation of succinate and the release of CO2. Most obviously, oxidation of the prime substrate proceeds via a highly oxidizing iron(IV) oxene intermediate.In a previous study, we showed that both phytanoyl-CoA and 3-methylhexadecanoyl-CoA are efficiently hydroxylated by recombinant PAHX, whereas in vitro, no activity toward 2- or 4-methyl-branched acyl-CoA esters or toward long and very long straight-chain acyl-CoA esters could be detected (9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar). Normally, reactions catalyzed by dioxygenases are highly stereospecific, occurring with retention of the configuration (17Prescott A.G. Lloyd M.D. The iron(II) and 2-oxoacid-dependent dioxygenases and their role in metabolism.Nat. Prod. Rep. 2000; 17: 367-383Crossref PubMed Scopus (164) Google Scholar). However, we previously demonstrated that α-oxidation of phytanic acid is not stereoselective with regard to the 3-methyl branch, although closer analysis revealed that the orientation of the β-substituent determines the stereochemistry of the α-hydroxylation (9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar, 18Croes K. Casteels M. Dieudaide-Noubhani M. Mannaerts G.P. Van Veldhoven P.P. Stereochemistry of the α-oxidation of 3-methyl-branched fatty acids in rat liver.J. Lipid Res. 1999; 40: 601-609Abstract Full Text Full Text PDF PubMed Google Scholar). Taken together, these observations would indicate that the enzyme recognizes only the branching point, not the length of the branch, and that the catalytic pocket allows access of both isomers. Therefore, we investigated the substrate spectrum of PAHX with regard to the length of both the acyl chain and the branch at position 3.MATERIALS AND METHODSMaterials4-Phenyl-2-butanol (99%), 2-undecanol (99%), and methanesulfonyl chloride were from Acros. 3-Phenylbutyric acid (98%), 3-methylpentanoic acid (98%), 2-methylhexanoic acid (99%), citronellic acid (3,7-dimethyloct-6-enoic acid; 98%), 2-octanol (99%), 2-decanol (98%), pyridine, and borane-methyl sulfide were purchased from Aldrich. 4-Methylnonanoic acid (97%) and 4-decanol (97%) were obtained from Avocado. 5-Nonanol (99.5%), diethyl malonate, 1,1′-carbonyldiimidazole, and 2,2-dimethoxypropane were from Fluka. 3-Nonanol (97%) was from Merck.Synthesis of branched-chain fatty acidsUnless otherwise mentioned, all branched fatty acids (and derivatives) were synthesized as racemic mixtures. 5-Phenyl-3-methylpentanoic acid, 3-methylnonanoic acid, 3-methylundecanoic acid, 3-methyldodecanoic acid, 3-ethylnonanoic acid, 3-propylnonanoic acid, and 3-butylheptanoic acid were obtained by chain elongation of, respectively, 4-phenyl-2-butanol, 2-octanol, 2-decanol, 2-undecanol, 3-nonanol, 4-decanol, and 5-nonanol with diethyl malonate, essentially as described by Spener and Mangold (19Spener F. Mangold H.K. Reactions of aliphatic methanesulfonates. VII. Chain elongation by two methylene groups.Chem. Phys. Lipids. 1973; 11: 215-218Crossref Scopus (24) Google Scholar). Briefly, diethyl malonate was alkylated with the respective mesylates (prepared from the corresponding alcohols and methanesulfonyl chloride in pyridine) in a ratio of 1:1.15 (v/v). The diethylesters were saponified with 2N NaOH in 10% aqueous ethanol and subsequently extracted under acidic conditions to recover the corresponding dicarboxylic acids. Decarboxylation was performed by pyrolysis at 165°C (120 min) in ethylene glycol. After cooling, water was added and the resulting fatty acids were extracted into chloroform. 5-Phenyl-3-methylpentanoic acid, 3-methylundecanoic acid, 3-methyldodecanoic acid, 3-ethylnonanoic acid, 3-propylnonanoic acid, and 3-butylheptanoic acid were further purified over Sep-Pak NH2 cartridges and eluted with increasing concentrations of acetic acid in diethylether (2–4%). Purity of the end products was checked by TLC (Silicagel 60, Merck; hexane-diethylether-acetic acid, 60:40:1, v/v/v) after spraying with bromocresol green. Rf values were 0.82 (3-methylundecanoic acid), 0.81 (3-ethylnonanoic acid), 0.79 (3-propylnonanoic acid), and 0.83 (3-butylheptanoic acid), and yields varied between 15% and 30%.3-Methylheptanoic acid was prepared from 2-methylhexanoic acid, which was first transformed in the corresponding alcohol in tetrahydrofuran (THF) with borane-methylsulfide. The mesylate (generated as described above) was further converted into 2-methylhexyl-1-nitrile in a substitution reaction with KCN. Alkaline oxidation and hydrolysis of the nitrile finally yielded 3-methylheptanoic acid, which was dried in the presence of triethylamine. Purity was checked by TLC (Silicagel 60, Merck; hexane-diethylether-acetic acid, 80:20:1, v/v/v; Rf 0.57).Synthesis of branched-chain fatty acyl-CoA estersThe CoA esters of 3-methylhexadecanoic acid and its 3R- and 3S-isomers (9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar) and of 2-methylhexadecanoic acid (20Van Veldhoven P.P. Vanhove G. Vanhoutte F. Dacremont G. Parmentier G. Eyssen H.J. Mannaerts G.P. Identification and purification of a peroxisomal branched fatty acyl-CoA oxidase.J. Biol. Chem. 1991; 266: 24676-24683Abstract Full Text PDF PubMed Google Scholar) were prepared as described previously. Prior to conversion into the corresponding acyl-CoA esters, 3-phenylbutyric acid, 3-methylpentanoic acid, 5-phenyl-3-methylpentanoic acid, 3,7-dimethyloct-6-enoic acid, 3-methylnonanoic acid, 4-methylnonanoic acid, 3-methyldodecanoic acid, 3-methylundecanoic acid, 3-ethylnonanoic acid, 3-propylnonanoic acid, and 3-butylheptanoic acid (250 μmol) were activated with 1.2-fold molar excess 1,1′-carbonyldiimidazole in tetrahydrofuran (for the activation of 4-methylnonanoic acid, dimethoxypropane was added as a moisture scavenger). The activation of 3-methylheptanoate-triethylamine salt was performed in the presence of an equimolar concentration of 2,6-dichlorobenzoic acid in respect to triethylamine. The imidazole derivatives were subsequently dried and dissolved in THF. CoA esterification was initiated at room temperature by the addition of an aqueous solution of CoA-Li3 in 0.5 M NaHCO3 (pH 8.5); the reaction was allowed to proceed at 4°C. In order to purify 5-phenyl-3-methylpentanoyl-CoA, 3-methylnonanoyl-CoA, 4-methylnonanoyl-CoA, 3-methyldodecanoyl-CoA, 3-methylundecanoyl-CoA, 3-ethylnonanoyl-CoA, 3-propylnonanoyl-CoA, and 3-butylheptanoyl-CoA, each mixture containing ∼50 μmol of CoA/CoA ester, were lyophilized, taken up in 10 ml of water, and acidified with perchloric acid. The precipitated acyl-CoAs, collected by centrifugation, were washed once with 0.8% perchloric acid and once with acetone. The residues were dissolved in 5 ml 0.1 M NaHCO3, applied to RP-C18-cartridges, and eluted with increasing concentrations of methanol in water. To purify 3-methylheptanoyl-CoA, 3-methylpentanoyl-CoA, 3-phenylbutyryl-CoA, and 3,7-dimethyloct-6-enoyl-CoA, the reaction mixtures were lyophilized after adding 0.25 ml 1 M ammonium acetate (pH 5). Subsequently, the residue was dissolved in 1 M ammonium acetate (pH 5) and applied to an RP-C18-cartridge. The CoA esters were eluted from the column with increasing concentrations of methanol in 0.5 M ammonium acetate (pH 5).The following yields (related to CoA input) were obtained: 3-methylheptanoyl-CoA (23.8 mg; 53%), 3-phenylbutyryl-CoA (18.4 mg; 39%), 3-methylpentanoyl-CoA (18 mg; 40%), 3,7-dimethyloct-6-enoyl-CoA (10.1 mg; 21%), 3-methylundecanoyl-CoA (39.6 mg; 80%), 3-ethylnonanoyl-CoA (21 mg; 43%), 3-propylnonanoyl-CoA (28.3 mg; 57%), and 3-butylheptanoyl-CoA (14.7 mg; 30%).Generation and purification of recombinant human PAHXRecombinant mature human PAHX was generated and purified as described previously (9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar), with slight modifications. Briefly, the enzyme was purified from 2 l of culture containing Top10F′ cells (Invitrogen) expressing His-tagged PAHX, 4 h post induction with isopropyl-1-thio-β-d-galactopyranoside. After centrifugation (Kontron A 6.14; 10 min at 8,800 rpm), the cell pellet was dissolved in 40 ml of 20 mM Na-phosphate buffer (pH 7.4) containing 0.5 M NaCl, 10 mM imidazole, and protease inhibitors. Following sonication of the cell suspension and subsequent centrifugation (Kontron A 8.24; 15 min at 11,000 rpm), the supernatant was applied on Ni-NTA (Qiagen) and the poly-His-tagged protein was eluted from the column with 20 mM Na-phosphate buffer (pH 7.4) containing 0.5 M NaCl and 250 mM imidazole. Yield was ∼1 mg of protein, and specific activities varied between 216 and 236 nmol/min−1/(mg protein)−1 (50 μM 3-methylhexadecanoyl-CoA as substrate; standard conditions).Hydroxylation of acyl-CoA esters by recombinant human PAHXHydroxylation reactions were performed as described previously (9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar), with some modifications. Incubations (37°C) were started by the addition of 5 μg of purified recombinant human PAHX, appropriately diluted in 50 μl of 20 mM Na-phosphate buffer (pH 7.4) containing 0.5 M NaCl and 250 mM imidazole, to 200 μl of reaction medium. Final concentrations were 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 12.5 μM defatted BSA, 4 mM GTP, 0.2 mM CoA, 2.4 mM MgCl2, 0.1 mM FeCl2, 10 mM l-ascorbate, and 3 mM 2-oxoglutarate (referred to as standard conditions). Substrate concentrations were 50 μM, unless otherwise mentioned. Reactions were stopped after 10 min by adding 25 μl of 1 N H2SO4. After addition of 8 nmol of internal standard (octanoyl-CoA or hexadecanoyl-CoA, depending on the substrate used), the samples were extracted with 1,200 μl of isopropanol-heptane, 4:1 (v/v). Subsequently, the supernatant was dried under N2 at 40°C after addition of 20 μl of 2% (v/v) reduced Triton X-100 and the samples were finally reconstituted in 100 μl of buffer A [CH3CN-H2O-0.25 M NH4OAc (pH 5.0) 10:60:20, v/v/v]. An aliquot (∼90 μl) was injected onto a NovaPak C18 column (Waters; 3.9 × 150 mm; 60 Å; 4 μm), and the CoA esters were eluted with increasing concentrations of buffer B [CH3CN-H2O-0.25 M NH4OAc (pH 5.0), 80:10:10, v/v/v] in buffer A. Flow rate was 0.8 ml/min, and effluents were monitored by using an on-line UV detector (Waters 486) set at 258 nm (see Fig. 1).RESULTS AND DISCUSSIONKinetic analysis of PAHX-catalyzed conversion of 3-methylacyl-CoA estersWhen the activity of purified recombinant human PAHX toward racemic 3-methylhexadecanoyl-CoA was tested at increasing substrate concentrations and a molar substrate-albumin ratio of 2, a plateau was reached from 100 μM onwards (Fig. 2A);an apparent Km of 40.8 μM was calculated. Figure 2B further shows that for a substrate concentration of 50 μM, the reaction was linear for up to 10 min.Fig. 2Functional characterization of recombinant human PAHX. A: Substrate dependence of recombinant human PAHX. Hydroxylase activity was measured at increasing concentrations of 3-methylhexadecanoyl-CoA and a molar substrate:albumin ratio of 2. Results are presented as nmol product generated in the assay. Inset: Lineweaver-Burk transformation of the obtained data. B: Time curve. Conversion of 3-methylhexadecanoyl-CoA was measured at different time intervals with a substrate concentration of 50 μM and a molar substrate:albumin ratio of 4. Final assay volume was 500 μl. C: Dependence of the hydroxylation reaction on albumin. The hydroxylation of 50 μM 3-methylhexadecanoyl-CoA (triangle), 3-methylundecanoyl-CoA (square) and 3-methylnonanoyl-CoA (circle) was assayed in the presence of increasing substrate:albumin ratios. D: Dependence of the hydroxylation of 3-methylhexadecanoyl-CoA on β-cyclodextrin. The hydroxylation of 50 μM 3-methylhexadecanoyl-CoA was measured in the absence of albumin at increasing substrate-β-cyclodextrin ratios.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When assaying the hydroxylation of 3-methylhexadecanoyl-CoA (50 μM) at increasing substrate-albumin ratios (ν), a plateau was reached from a ratio of 4 onwards (Fig. 2C). Interestingly, at this substrate concentration, no hydroxylation of 3-methylhexadecanoyl-CoA could be detected in the absence of albumin (ν = ∞ ) (Fig. 2C). However, upon lowering the substrate concentration in the absence of albumin, the bulk of 3-methylhexadecanoyl-CoA was converted to its 2-hydroxy intermediate (100% conversion was measured for concentrations up to 5 μM; at a final concentration of 10 μM, 63.5% of the substrate was hydroxylated; at a final concentration of 25 μM, only 44%; data not shown). The conversion of shorter 3-methylacyl-CoA esters (50 μM 3-methylnonanoyl-CoA or 3-methyldodecanoyl-CoA) was much less dependent on the presence of albumin (Fig. 2C).Taken together, these observations suggest that the enzyme cannot display any activity when the substrate is presented as a micelle. The critical micellar concentration (CMC) of 3-methylhexadecanoyl-CoA is likely comparable to the CMC of palmitoyl- and stearyl-CoA. As for the former compound, CMC values of 30 μM to 60 μM have been reported (21Constantinides P.P. Steim J.M. Physical properties of fatty acyl-CoA. Critical micelle concentrations and micellar size and shape.J. Biol. Chem. 1985; 260: 7573-7580Abstract Full Text PDF PubMed Google Scholar, 22Smith R.H. Powell G.L. The critical micelle concentration of some physiologically important fatty acyl-coenzyme coenzyme As as a function of chain length.Arch. Biochem. Biophys. 1986; 244: 357-360Crossref PubMed Scopus (43) Google Scholar, 23Powell G.L. Grothusen J.R. Zimmerman J.K. Evans C.A. Fish W.W. A re-examination of some properties of fatty acyl-CoA micelles.J. Biol. Chem. 1981; 256: 12740-12747Abstract Full Text PDF PubMed Google Scholar), in all our experiments performed with 50 μM of 3-methylhexadecanoyl-CoA, the substrate might have been presented in micelles. Albumin, through binding the monomeric CoA-ester, will lower the amount of micelles, and hence stimulate the activity at substrate concentrations above the CMC. At low ν-ratios (ν = 1–3), almost all acyl-CoA is complexed to BSA. Hence, the lower activity under these conditions would further indicate that the enzyme is also not able to act on albumin-bound substrate. At low substrate concentrations, no micelles are formed, explaining the high conversion rates, even in the absence of BSA. For shorter 3-methylacyl-CoA esters, which possess a higher CMC, the hydroxylation rates at 50 μM are less influenced by the concentration of albumin (Fig. 2C).During this work, Mukherji et al. (24Mukherji M. Kershaw N.J. Schofield C.J. Wierzbicki A.S. Lloyd M.D. Utilization of sterol carrier protein-2 by phytanoyl-CoA 2-hydroxylase in the peroxisomal alpha oxidation of phytanic acid.Chem. Biol. 2002; 9: 597-605Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) reported on the use of β-cyclodextrin as required for PHAX measurements as a “solubilizing agent.” As acyl-CoA esters are detergents themselves, β-cyclodextrin cannot act as a substrate-solubilizing tool. In the absence of albumin, a beneficial effect of β-cyclodextrin was observed, but rather high amounts were required, i.e., a 2- to 8-fold molar excess over substrate (Fig. 2D). At these concentrations, cyclodextrin will likely capture acyl-CoAs, thereby lowering the amount of micellar substrate. A similar explanation might account for the stimulatory effect of SCP2, seen at high substrate concentrations by Mukherji et al. (24Mukherji M. Kershaw N.J. Schofield C.J. Wierzbicki A.S. Lloyd M.D. Utilization of sterol carrier protein-2 by phytanoyl-CoA 2-hydroxylase in the peroxisomal alpha oxidation of phytanic acid.Chem. Biol. 2002; 9: 597-605Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). On the basis of this observation, these authors suggested that in vivo SCP2 could act as a “solubilizing agent” and that the in vivo substrates for PAHX may be SCP2 complexes. The ARD-like symptoms and biochemical alterations observed in SCP2-deficient mice (25Seedorf U. Raabe M. Ellinghaus P. Kannenberg F. Fobker M. Engel T. Denis S. Wouters F. Wirtz K.W. Wanders R.J. Maeda N. Assmann G. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-X function.Genes Dev. 1998; 12: 1189-1201Crossref PubMed Scopus (243) Google Scholar) support this contention. However, a beneficial effect of SCP2 on PAHX activity is seen only at high substrate concentrations, which might never be reached under physiological conditions, rendering this hypothesis rather implausible.Both a CoA moiety and a branch at position 3 are required for PAHX-dependent hydroxylationAlthough PAHX efficiently catalyzes the hydroxylation of phytanoyl-CoA and its chemical substitute, 3-methylhexadecanoyl-CoA, no PAHX-dependent hydroxylation of 3-methylhexadecanoic acid could be observed. This indicates that in α-oxidation, the activation step, which precedes the hydroxylation step (9Croes K. Foulon V. Casteels M. Van Veldhoven P.P. Mannaerts G.P. Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-CoAs and requirement for GTP or ATP and Mg2+ in addition to its known hydroxylation cofactors.J. Lipid Res. 2000; 41: 629-636Abstract Full Text Full Text PDF PubMed Google Scholar), is necessary for PAHX-dependent hydroxylation. In contrast to the data reported by Mukherji et al. (24Mukherji M. Kershaw N.J. Schofield C.J. Wierzbicki A.S. Lloyd M.D. Utilization of sterol carrier protein-2 by phytanoyl-CoA 2-hydroxylase in the peroxisomal alpha oxidation of phytanic acid." @default.
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