The aim of this investigation is to assess whether long-chain fatty acids can be a substrate for omega-oxidation and the subsequent beta-oxidation to produce medium-chain dicarboxylic acids normally found in urine. Isolated rat liver 10,000 g supernatant and pellet fractions were used as the source of enzymes. The metabolism of palmitate was studied using [1,2,3,4-13C4]hexadecanoic acid as tracer. Selected ion monitoring mass spectrometry was utilized for the determination of isotope enrichments in precursor and products. Palmitate was found to be a good substrate for omega-oxidation; the rate was only slightly slower than decanoate. The beta-oxidation of [1,2,3,4-13C4]hexadecanedioic acid yielded labeled adipic, suberic, and sebacic acids. Isotope distribution in these dicarboxylic acids consisted mostly of unlabeled molecules (M + 0) and molecules labeled with four 13C (M + 4), in agreement with a beta-oxidation initiated equally from both carboxyl ends of the precursor. Significant enrichments (1-8%) with only two 13C labels (M + 2) indicate a partial bidirectional beta-oxidation. The direct metabolic conversion of hexadecanedioate to succinate was documented by the significant enrichment (1.40-1.90%) in M + 4 of succinate. These data indicate that long-chain fatty acids can be a substrate for the production of medium-chain dicarboxylates and the eventual direct conversion to succinate.
Biosynthesis of Medium-Chain ω-Hydroxy Fatty Acids by AlkBGT of Pseudomonas putida GPo1 With Native FadL in Engineered Escherichia coli
