A dual function alpha-dioxygenase-peroxidase and NAD(+) oxidoreductase active enzyme from germinating pea rationalizing alpha-oxidation of fatty acids in plants.
An enzyme with fatty acid alpha-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 alpha-oxidation of palmitic acid incubated at 0 degrees C with the purified alpha-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 alpha-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 alpha-oxidation in plants.
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.
A
A
Saffert
J
J
Hartmann-Schreier
A
A
Schön
P
P
Schreier
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism*; D018525:Germination; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology* / Q000502:physiology; D010544:Peroxidases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D005227:Fatty Acids; D009243:NAD; D019308:Palmitic Acid; D010088:Oxidoreductases; D010544:Peroxidases; D010105:Oxygenases