A G316A Mutation of Manganese Lipoxygenase Augments Hydroperoxide Isomerase Activity

Abstract

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.

Catalytic Convergence of Manganese and Iron Lipoxygenases by Replacement of a Single Amino Acid

“…Oxygen Access-As previously reported, G332A⅐13R-MnLOX augmented the hydroperoxide isomerase activity and the formation of epoxy alcohols from 13R-HPOTrE (38). We found that L336F⅐13R-MnLOX also possessed prominent hydroperoxide isomerase activity.…”

“…This is also in analogy with G332A, which only slightly increased the oxygenation at C-9 (38). K m for 18:3n-3 remained in the low M range for L336F (Table 2).…”

“…S1). Sequential N-terminal sequencing suggested that mini 13R-MnLOX was mainly formed by cleavage between Thr 37 and Thr 38 as judged from five amino acids in correct sequence order from this point (data not shown). This corresponded to a protein of 580 amino acids with a calculated molecular mass of 63.8 kDa.…”

“…G332A⅐13R-MnLOX has previously been found to enhance the dehydration of 13R-HPOTrE (38). A notable difference a Relative data from analysis of end products from incubation with 13R-MnLOX and its mutants with 100 M substrate in 0.1 M NaBO 3 (pH 9.0).…”

“…A few LOXs, including 9S-MnLOX, deviate from this rule of R/S stereospecificity (35,36). The Gly/Ala position also influences oxygen access and the hydroperoxide isomerase activities of eLOX-3 and 13R-MnLOX (37,38).…”