Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino acid.

Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE

Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology
Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology

Amino Acids/*chemistry ; Iron/*chemistry ; Lipoxygenase/*chemistry ; Lipoxygenase/*genetics ; Manganese/*chemistry; Amino Acid Sequence ; Animals ; Anthozoa ; Arachidonate Lipoxygenases/chemistry ; Catalysis ; Chromatography, High Pressure Liquid/methods ; Humans ; Hydrogen/chemistry ; Kinetics ; Mass Spectrometry/methods ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Oxygen/chemistry ; Pichia/metabolism ; Sequence Homology, Amino Acid ; Glycine max/enzymology

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.

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0 (Amino Acids)
42Z2K6ZL8P (Manganese)
7YNJ3PO35Z (Hydrogen)
E1UOL152H7 (Iron)
EC 1.13.11.- (Arachidonate Lipoxygenases)
EC 1.13.11.12 (Lipoxygenase)
EC 1.13.11.40 (arachidonate 8-lipoxygenase)
S88TT14065 (Oxygen)

Date Created: 20120724 Date Completed: 20121204 Latest Revision: 20231213

20240829

PMC3442510

10.1074/jbc.M112.364331

22822060