Plant Physiol, August 2000, Vol. 123, pp. 1545-1552

A Dual Function -Dioxygenase-Peroxidase and NAD⁺ Oxidoreductase Active Enzyme from Germinating Pea Rationalizing -Oxidation of Fatty Acids in Plants^1,2

Institutes of Pharmacy and Food Chemistry (A. Saffert, J.H.-S., P.S.) and Biochemistry (A. Schön), University of Würzburg, Am Hubland, D-97074 Würzburg, Germany

An enzyme with fatty acid -oxidation activity (49 nkat mg¹; 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 -oxidation of palmitic acid incubated at 0°C with the purified -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 -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 -oxidation in plants.