title: Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino acid.
abstract: Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (∼80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ∼100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.
authors: Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors: Anneli
authors: A
authors: Wennman
authors: Fredrik
authors: F
authors: Jernerén
authors: Mats
authors: M
authors: Hamberg
authors: Ernst H
authors: EH
authors: Oliw
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D037421:Anthozoa; D001091:Arachidonate Lipoxygenases / Q000737:chemistry; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics*; D008345:Manganese / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology
chemical_list: D000596:Amino Acids; D008345:Manganese; D006859:Hydrogen; D007501:Iron; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase; C054192:arachidonate 8-lipoxygenase; D010100:Oxygen