Free fatty acohols have been established as lipid components of E. coli K-12. Using combined gas liquid chromatography-mass spectrometry, the major alcohols in aerobically grown cells were identified as 1-tetradecanol (18%), 1hexadecanol (28%), 1-octadecanol (14%), and 2 -p e n t a d e c a n o l (27%). Small amounts of 1-hexadecenol (3%), 2-tridecanol (8%), and 2-tetradecanol (1.5%) were also detected. Analysis of anaerobically grown cells has shown a selective decrease of the secondary alcohols. 2-Pentadecanol was present as only 7% of the total alcohol fraction, and only traces of 2-tridecanol and 2-tetradecanol were found. The major alcohols in anaerobic cells were 1-tetradecanol, 1-pentadecanol, 1-hexadecenol and 1-hexadecanol. The above observations strongly suggest two pathways for the synthesis of fatty alcohols in E. coli..One pathway synthesizes the primary alcohols and does not require molecular oxygen, and a separate pathway synthesizes the secondary alcohols and has a requirement for molecular oxygen.
In vivo studies have indicated that exogenous free fatty acids may serve as precursors of the free fatty alcohols ofEscherichia coli K‐12. Following disruption of the cells, the enzymatic activity capable of catalyzing the reduction of long chain fatty aldehydes to fatty alcohols was localized in the 100,000 x g supernatant fraction. Nicotinamide adenine dinucleotide phosphate, reduced form, was the required cofactor. The product of the reaction was characterized rigorously as 1‐hexadecanol when hexadecanal was the substrate. Three independent, but complementary, assay methods were developed to assay the aldehyde reductase activity. By employing these methods, an equivalence between nicotinamide adenine dinucleotide phosphate, reduced form, oxidation and 1‐hexadecanol synthesis was established. Two protein fractions catalyzing the reduction of fatty aldehydes to fatty alcohols were detected in the 100,000 x g supernatant fraction following ammonium sulfate fractionation and diethylaminoethyl‐cellulose chromatography. Enzymatic activity (70%) applied to the diethylamino‐ethyl‐cellulose column was eluted at a phosphate concentration of 0.115 M. The remaining 30% was eluted at a concentration of 0.23 M. Following sephadex chromatography, it was observed that the enzyme eluting at 0.115 M phosphate had an apparent mol wt of 250,000 Daltons while that eluting at 0.23 M had an apparent mol wt of 62,000 Daltons. The enzymes were similar with respect to substrate specificity, pH optima, ionic strength optima, and stability with respect to thiol inhibitors, suggesting different sized aggregates of similar subunits.
HeLa cells exposed to trace amounts of pentadecan-2-one showed changes in metabolism of 1(-14)C-palmitate. These changes consisted of an increased incorporation of radioactivity into the triglycerides and free fatty acids and a decreased 14C incorporation into the ether moiety of alk-1-enyl acyl phosphoglycerides. Chemical analysis of the several lipid fractions showed a threefold increase in triglyceride content but no change in the amount of alk-1-enyl acyl or diacyl phosphoglycerides in the treated cells. Pentadecan-2-one added to the culture medium apparently gains entrance to the cell since both pentadecan-2-one and pentadecan-2-ol were detected in the ketone-treated cells and their culture medium.
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