A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols.
Lipoxygenases with R stereospecificity have a conserved Gly residue, whereas (S)-lipoxygenases have an Ala residue. Site-directed mutagenesis has shown that these residues control position and S/R stereospecificity of oxygenation. Recombinant Mn-LO was expressed in Pichia pastoris, and its conserved Gly-316 residue was mutated to Ala, Ser, Val, and Thr. The G316A mutant was catalytically active. We compared the catalytic properties of Mn-LO and the G316A mutant with 17:3n-3, 18:2n-6, 18:3n-3, and 19:3n-3 as substrates. Increasing the fatty acid chain length from C17 to C19 shifted the oxygenation by Mn-LO from the n-6 toward the n-8 carbon. The G316A mutant increased the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 1-2% to 7-11%). The most striking effect of the G316A mutant was a 2-, 7-, and 15-fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. The n-3 double bond was essential. An experiment under an oxygen-18 atmosphere showed that both oxygen atoms were retained in the epoxyalcohols. (R)-Hydroperoxides at n-6 of C17:3, 18:3, and 19:3 were transformed 5 times faster than S stereoisomers. The G316A mutant converted (13R)-hydroperoxylinolenic acid to 13-ketolinolenic acid (with an apparent K(m) of 0.01 mm) and to epoxyalcohols (viz. erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products). A reducing lipoxygenase inhibitor stimulated the hydroperoxide isomerase activity, whereas a suicide-type lipoxygenase inhibitor reduced this activity. The n-3 double bond also appeared to influence the anaerobic formation of epoxyalcohols by Mn-LO, since 18:2n-6 and 18:3n-3 yielded different profiles of epoxyalcohols. Our results suggest that the G316A mutant augmented the hydroperoxide isomerase activity by positioning the hydroperoxy group at the n-6 carbon of n-3 fatty acids closer to the reduced catalytic metal.
Cristea
Mirela
M
Department of Pharmaceutical Biosciences, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden.
Oliw
Ernst H
EH
D000438:Alcohols / Q000378:metabolism*; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000502:physiology; D004852:Epoxy Compounds / Q000378:metabolism*; D015525:Fatty Acids, Omega-3 / Q000378:metabolism; D006427:Hemin / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D008054:Lipid Peroxides / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010843:Pichia; D013237:Stereoisomerism; D013379:Substrate Specificity; D017962:alpha-Linolenic Acid / Q000378:metabolism
D000438:Alcohols; D004852:Epoxy Compounds; D015525:Fatty Acids, Omega-3; D008054:Lipid Peroxides; D017962:alpha-Linolenic Acid; D006427:Hemin; C112608:manganese lipoxygenase; D008084:Lipoxygenase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase; D010100:Oxygen