An enzyme with fatty acid ␣-oxidation activity (49 nkat mg Ϫ1 ; 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 ϩ 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 ϩ oxidoreductase (50-kD unit) we provided novel data to rationalize all steps of the classical scheme of ␣-oxidation in plants.Fatty acid hydroperoxides are reactive intermediates in the oxylipin pathways of fatty acid oxygenation in plants and fungi
The substrate selectivities of the α oxidation of saturated, unsaturated, and heteroatom-containing
(oxygen, sulfur) carboxylic acids 1 by the enzyme extract of peas (Pisum sativum) indicate that this
biotransformation proceeds highly enantioselectively. For the first time, the synthesis of optically pure 2-hydroxy
acids 2 has been achieved on the semipreparative scale (1 mmol) by α hydroxylation of long-chain carboxylic
acids with molecular oxygen, catalyzed by the α oxidase of peas. For derivatives with sulfur atom in the
chain, no sulfoxidation is observed. The functionalities (carbon double and triple bonds, oxygen, and sulfur
atoms) must be at least three carbon atoms away from the carboxylic acid group to achieve efficient asymmetric
hydroxylation. The absolute configuration of the 2-hydroxy acids 2 was assigned by comparison of the gas-chromatographic data with that of authentic reference compounds and by application of the exciton-coupled-circular-dichroism (ECCD) method. This unprecedented asymmetric biocatalytic methodology should be
valuable for the preparation of enantiomerically pure (R)-2-hydroxy acids.
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