A dual func tion α-dioxygenase-peroxidase and NAD+ oxidoreductase active enzyme from germinating Pea rationalizing α-oxidation of fatty acids in plants

Abstract

An enzyme with fatty acid α-oxidation activity (49 nkat mg-1; substrate: lauric acid) was purified from germinating pea (Pisum sativum) by a five-step procedure to apparent homogeneity. The purified protein was found to be a 230-kD oligomer with two dominant subunits, i.e. a 50-kD subunit with NAD+ oxidoreductase activity and a 70-kD subunit, homolog to a pathogen-induced oxygenase, which in turn shows significant homology to animal cyclooxygenase. On-line liquid chromatography-electrospray ionization-tandem mass spectrometry revealed rapid α-oxidation of palmitic acid incubated at 0°C with the purified α-oxidation enzyme, leading to (R)-2-hydroperoxypalmitic acid as the major product together with (R)-2-hydroxypalmitic acid, 1-pentadecanal, and pentadecanoic acid. Inherent peroxidase activity of the 70-kD fraction decreased the amount of the (R)-2-hydroperoxy product rapidly and increased the level of (R)-2-hydroxypalmitic acid. Incubations at room temperature accelerated the decline toward the chain-shortened aldehyde. With the identification of the dual function α-dioxygenase-peroxidase (70-kD unit) and the related NAD+ oxidoreductase (50-kD unit) we provided novel data to rationalize all steps of the classical scheme of α-oxidation in plants.