Biosynthesis of S-methyl-N-oleoylmercaptoethylamide from oleoyl coenzyme A and S-adenosylmethionine.

Abstract

Addition of oleoyl-CoA to incubations containing rat lung membranes and S-adenosyl [methyl-3H]methionine resulted in the formation of a previously unidentified nonpolar methylated lipid. The product was formed enzymatically, with an apparent Km for S-adenosylmethionine (AdoMet) of about 0.3 microM and half-maximal activity using about 0.1 mM oleoyl-CoA. Activity was highest in microsomes but present in other membranous fractions, including plasma membranes from mature human erythrocytes. Intact red blood cells formed the nonpolar methylated lipid intracellularly upon incubation with [methyl-3H]methionine and oleoyl-CoA. Product formation differed among membranes from various tissues. The nonpolar methylated lipid was analyzed by TLC, high performance liquid chromatography, and gas chromatography with radiodetection. It was identified as S-methyl-N-oleoylmercaptoethylamide by gas chromatography-mass spectrometry. Products obtained from oleoyl-CoA or palmitoyl-CoA, incubated with nonradioactive or [methyl-14C]AdoMet, were compared using electron impact and/or chemical ionization mass spectrometry. Inferred structures were confirmed using authentic standards. The methylated product was apparently formed by tissue as follows: (a) cleavage of oleoyl-CoA by an amidase to form S-oleoylmercaptoethylamine; (b) spontaneous rearrangement to form N-oleoylmercaptoethylamide; and (c) enzymatic methylation of the free thiol by AdoMet. Participation of the amidase was suggested by the biosynthesis of the amide (free thiol) using [1-14C]oleoyl-CoA.