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

• Published in: Plant physiology (Bethesda) 2000-08, Vol.123 (4), p.1545-1551
• Main Authors: Alexander Saffert, Jenny Hartmann-Schreier, Astrid Schön, Schreier, Peter
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Aldehydes; Amino Acid Sequence; Amino acids; Biochemical Processes and Macromolecular Structures; Biological and medical sciences; Chromatography, High Pressure Liquid; Economic plant physiology; Electrophoresis, Polyacrylamide Gel; Enzymes; Fatty acids; Fatty Acids - metabolism; Flow velocity; Fundamental and applied biological sciences. Psychology; Gels; Germination; Ionization; Lipids; Liquid chromatography; Mass Spectrometry; Metabolism; Molecular Sequence Data; NAD - metabolism; Nitrogen metabolism and other ones (excepting carbon metabolism); Nutrition. Photosynthesis. Respiration. Metabolism; Oxidation; Oxidation-Reduction; Oxidoreductases - metabolism; Oxygenases - metabolism; Palmitic Acid - metabolism; Peas; Peroxidases - metabolism; Pisum sativum - enzymology; Pisum sativum - physiology; Plant physiology and development; Proteins; Sequence Homology, Amino Acid; Unsaturated fatty acids
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.123.4.1545

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.