Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Abstract

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via β-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor β-oxidized in mitochondria. We thus examined whether ω-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their ω-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 ± 0.5 and 1.46 ± 0.30 nmol product formed/min/mg protein, respectively (mean ± SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal ω-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed ω-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were Km = 55 μM and Vmax = 8.33 min−1, whereas those for 3-hydroxypalmitate were Km = 56.4 μM and Vmax = 14.2 min−1. CYP4F11 kinetic values resembled those of native microsomes, with Km = 53.5 μM and Vmax = 13.9 min−1 for 3-hydroxystearate and Km = 105.8 μM and Vmax = 70.6 min−1 for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to ω-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted ω-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid β-oxidation increases circulating 3-OHFA levels.

Keywords

• dicarboxylic acids
• ω-hydroxylation
• dihydroxy fatty acids
• cytochrome 450 enzymes