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.

FIGURE 1.

Overview of possible lipoxygenation positions of linoleic acid and partial alignment of 13R-MnLOX, sLOX-1, 8R-LOX, human 5-LOX, and rabbit 15-LOX covering an important region for regio- and stereospecificity. A, overview of hydrogen abstraction and oxygenation of 18:2n-6. Lipoxidation by sLOX-1 occurs by abstraction of the pro-S hydrogen at C-11 with antarafacial oxygen insertion at C-13 with formation of 13S-HPODE, whereas 9S- and 13R-MnLOX abstract the very same hydrogen but form 9S- and 13R-HPODE, respectively, as the main products (suprafacial oxygen insertion). B, partial amino acid sequences with the conserved determinant (Gly/Ala) for regio- and R/S stereospecificity and the Sloane determinant of regiospecificity (see Refs. , , , and 33) as marked by arrows. The catalytic importance of Leu³³⁶ and Phe³³⁷ of 13R-MnLOX was investigated in this report.

FIGURE 2.

SDS-PAGE analysis of recombinant mini 13R-MnLOX and chiral phase HPLC-MS/MS analysis of products formed by F337I·13R-MnLOX. A, SDS-PAGE analysis of mini 13R-MnLOX revealed extensive glycosylation (band at 95–110 kDa). B, oxidation of 18:2n-6 by F337I. Products in peaks I–IV of the top chromatogram were identified by MS/MS analysis as indicated by the selective ion chromatograms for 13- and 9-HODE (MS/MS analysis, m/z 295 → full scan). A small amount of racemic [¹³C₁₈]9- and 13-HODE was added to facilitate identification of stereoisomers. C, oxidation of 18:3n-3 by F337I·13R-MnLOX. Products in peaks I–IV were identified by MS/MS analysis as indicated in the selective ion chromatograms from MS/MS analysis (m/z 293 → full scan). The stereoisomers were resolved on Chiralcel OBH. TIC, total ion current.

FIGURE 3.

Chiral phase HPLC-MS/MS analysis of products formed by the L336G·13R-MnLOX and L336F·13R-MnLOX. A, oxidation of 18:2n-6 by L336G·13R-MnLOX. Products in peaks I–III were identified as indicated by the selective ion chromatograms (MS/MS analysis, m/z 295 → full scan) of 13- and 9-HODE. A small amount of racemic [¹³C₁₈]9- and 13-HODE was added to facilitate identification of stereoisomers. B, oxidation of 18:3n-3 by L336F·13R-MnLOX. The metabolites in peaks I–IV were identified by selective ion chromatograms as shown and by complete MS spectra. Top, total ion current (TIC). Shown below are characteristic ion chromatograms for 13-HOTrE, 9-HOTrE, and 11-HOTrE; the stereoisomers of 11-HOTrE were not resolved (Chiralcel OBH).

FIGURE 4.

UV analysis of products formed by 13R-MnLOX with replacements near the catalytic metal. A, oxidation of 18:2n-6 by L336F·13R-MnLOX and L336V·13R-MnLOX under normal atmosphere. B, oxidation of 18:3n-3 by L336F·13R-MnLOX under normal atmosphere and in buffer saturated with oxygen, which mainly reduced the kinetic lag time. The inset shows that the decline in UV absorbance at 237 nm is accompanied by an increase in UV absorbance at 280 nm due to dehydration of hydroperoxides to keto metabolites. C, oxidation of 18:2n-6 and 18:3n-3 and subsequent dehydration of formed 13R-HPOTrE by 13R-MnLOX and G332A·13R-MnLOX under normal atmosphere and in buffer saturated with oxygen to products with UV absorbance at 237 nm.

FIGURE 5.

Normal phase HPLC-MS/MS analysis of the oxidation of 18:1n-6 by 13-MnLOX, G332A·13R-MnLOX, and L336F·13R-MnLOX. A, formation of 13-hydroxyoctadecenoic acid (HOME) (11E) and 11-hydroxyoctadecenoic acid (12Z) by 13R-MnLOX (top trace) and G332A·13R-MnLOX (bottom trace). B, oxidation of 18:1n-6 by L336F·13R-MnLOX. The inset shows oxidation of 18:1n-6 by L336V. The different retention times in A and B are due to elution with 3 and 2% isopropyl alcohol in hexane.

FIGURE 6.

Overview of the catalytic effects of hydrophobic amino acid replacements near the metal center of 13R-MnLOX. L336F and G332A mainly increased the hydroperoxide isomerase activity. Replacement of Leu³³⁶ partly changed the oxidation of 18:3n-3 and 18:2n-6 from C-13 with retained R stereospecificity (>95%) toward C-9 with formation of 9S- and 9R-hydroperoxides. F337I changed the configuration of the 13-hydroperoxy metabolites of 18:3n-3 and 18:2n-6 from ∼100% R to ∼70% S.

FIGURE 7.

Comparison of the active sites of 13R-MnLOX and sLOX-1. A, the structure model of 13R-MnLOX (pink) in this figure was obtained with the aid of SWISS-MODEL using coral 8R-LOX (Protein Data Bank code 2fnq) as a template (52). PyMOL Molecular Graphics System (Schrödinger LLC) was used to align the model of 13R-MnLOX with sLOX-1 (Protein Data Bank code 1yge; cyan). The rectangle marks the active site, which is shown in detail in B. Linoleic acid is positioned in the active site with the pro-S hydrogen at C-11 close to the catalytic metal, the 12Z double bond close to Phe³³⁷/Ile⁵⁴⁷, and the ω-end toward the Sloane determinant. The model illustrates Gly³³², Leu³³⁶, Phe³³⁷, and Phe³⁴⁷ of 13R-MnLOX and the corresponding residues, Ala⁵⁴², Leu⁵⁴⁶, Ile⁵⁴⁷, and Phe⁵⁵⁷, of sLOX-1. Gly³³²/Ala⁵⁴² are in the Coffa-Brash determinant, and Phe³⁴⁷/Phe⁵⁵⁷ are in the Sloane determinant. C and D, schematic views of linoleic acid in the active sites of 13R-MnLOX (C) and sLOX-1 (D). Relative to the hydrogen abstraction at C-11, Phe³³⁷ of 13R-MnLOX and Ile⁵⁴⁷ of sLOX-1 may block antarafacial and suprafacial oxygen insertion at C-13, respectively.