A G316A Mutation of Manganese Lipoxygenase Augments Hydroperoxide Isomerase Activity

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 Km 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.

. Iron lipoxygenases occur in animals and plants, whereas manganese lipoxygenase has been demonstrated in Gaeumannomyces graminis, the take-all fungus of wheat (6), and homologous genes occur in the rice blast fungus, Magnaporthe grisea, and in Aspergillus fumigatus (XP_362938 and XP_746463, respectively, reported in GenBank TM , available on the World Wide Web at www.ncbi.nlm.nih.gov).
The three-dimensional protein structures are available for sLO-1 and -3, rabbit reticulocyte (15S)-LOX, and coral (8R)-LOX (24 -28). Single or sequential mutations of residues of the substrate binding cavity have been extensively studied, since they affect substrate positioning and may change the position specificity of the enzyme, from one end of the (1Z,4Z)-pentadiene to the other (29 -37). Coffa and Brash (38) observed that lipoxygenases, which form hydroperoxides with S chirality, have a conserved Ala residue (or, occasionally, a Ser residue), whereas human and mouse (12R)-LOXs and coral (8R)-LOX have a conserved Gly res-idue in the corresponding position ( Table 1). Mutation of Ala to Gly of two (S)-lipoxygenases (human (15S)-LOX-2 and mouse (8S)-LOX) altered the chirality of oxygenation to R and also the position specificity to the other end of the (1Z,4Z)-pentadiene, whereas the Gly to Ala mutation of two (R)-lipoxygenases (human (12R)-LOX and coral (8R)-LOX) ( Table 1) changed the position and chirality to S in the same fashion (38). These results have now been extended to mutations of sLO-1 and murine (12R)-LOX (37,39). The results could be explained by a model where the Gly and Ala residues change the substrate position relative to an oxygen access channel, leading to antarafacial oxygen insertion at either end of the (1Z,4Z)-pentadiene (40,41). This oxygen channel appears to be narrow, since Gly to Val mutations of murine and human (12R)-lipoxygenases and Ala to Val mutations of three (S)-lipoxygenases led to loss of enzymatic activity (37,39). Whether Ala/Gly also affects the hydroperoxidase activity of eLOX3 (Table 1) has not yet been reported.
Mn-LO differs from all iron lipoxygenases in one important aspect; Mn-LO catalyzes suprafacial oxygenation, whereas iron lipoxygenases have been repeatedly found to catalyze antarafacial oxygenation (2). An explanation of this phenomenon could be that the oxygen access channel to the reactive pentadiene differs between Mn-LO and other lipoxygenases.
The amino acid sequence of Mn-LO can be aligned with 23-27% identity to plant and to mammalian S-and R-lipoxygenases, and the metal ligands are conserved (4,5). Mn-LO also contains the conserved Gly-316 residue of (R)-lipoxygenases (Table 1). Mn-LO catalyzes R lipoxygenation, but the alignments do not suggest a higher degree of amino acid identity with (R)-lipoxygenases than with (S)-lipoxygenases. The preferred substrate of Mn-LO is ␣-linolenic acid, which is oxidized at the n-8 carbon to (11S)-HPOTrE and the n-6 carbon to (13R)-HPOTrE, and the latter accumulates as the end product (2,6). The oxygenation of the bis-allylic carbon and hydroperoxide migration from C-11 to C-13 constitute an unprecedented feature of this lipoxygenase. Whether chain shortening of 18:3n-3 to 17:3n-3 or chain elongation to 19:3n-3 affects the oxidations of the n-6 and n-8 carbons have not been investigated.
Our first aim was to study the catalytic importance of Gly-316. We hypothesized that the different oxygenation mechanism and possible structural differences in oxygen access channels between Mn-LO and iron lipoxygenases might be revealed by mutation of Gly-316 to Ala, Val, Ser, and Thr residues, since these mutations of other (R)-lipoxygenases have been investigated (37,38). Our second aim was to compare the oxygenation of 17:3n-3, 18:3n-3, 18:2n-6, and 19:3n-3 by Mn-LO and its mutants.
We report that mutation of Gly-316 to Ala in Mn-LO changed the oxygenation of 17:3n-3 and 18:3n-3 essentially as predicted by 41), although the effects were less marked than anticipated. The other three mutations were catalytically silent. We found that the G316A mutation increased the hydroperoxide isomerase activity of (R)-hydroperoxides at the n-6 position of 19:3n-3, 18:3n-3, and 17:3n-3. As a mechanism, we propose that the G316A mutation shifts the position of the hydroperoxide group at the n-6 carbon closer to the reduced catalytic metal and that an n-3 double bond is essential to provide necessary structural rigidity by interaction with -electrons (42). Our results suggest that the Gly/Ala residues at position 316 of Mn-LO control the position of the n-3 fatty acids in relation to the oxygen insertion channel and the distance of the hydroperoxide group at n-6 to the catalytic metal.
Expression and Site-directed Mutagenesis-Recombinant Mn-LO was expressed in P. pastoris and secreted into the growth medium (buffered minimal methanol) using an expression construct with the alcohol oxidase promotor (pPICZ␣A-Mn-LO-602) and with the native secretion signal replaced with the yeast secretion ␣-signal, as described (5). Site-directed mutagenesis of pPICZ␣A-Mn-LO-602 was performed with oligonucleotides of 42 nucleotides and Pfu polymerase as described (5). The PCR mixture was treated with DpnI for 3 h, checked for amplification of the target plasmid by agarose gel electrophoresis, and used to transform E. coli by electroporation. Plasmid DNA was isolated and sequenced. Pichia cells were transformed as described (5), and recombi- nants were selected by Zeocin resistance (5). Genomic DNA was isolated from resistant colonies and screened for incorporation of the Mn-LO construct by PCR (forward, 5Ј-TGGATCTGACGTACACGCCCCTCGA; reverse, 5Ј-ATCCAGTTCTGTAGCTATAGTCG; annealing at 55°C) with equipment as described (5). We also confirmed by sequencing that the Pichia colonies used for expression contained the construct with the desired mutation. Enzyme Purification-Mn-LO was purified to homogeneity as described (5). Mn-LO G316A, G316S, G316V, and G316T were purified from the buffered minimal methanol medium (0.3-0.5 liters of Pichia cultures in baffled flasks). After 5-6 days of methanol induction, the A 600 reached ϳ15, and the cells were precipitated by centrifugation. Ammonium sulfate was added to the supernatant to 0.6 M, and the pH was adjusted to 7.0 (10 M NaOH). After centrifugation, the supernatant was loaded on a phenyl-Sepharose column (6). Bound proteins were eluted with low salt buffer and concentrated to ϳ1 ml by diafiltration. The expression of the recombinant proteins was checked by SDS-PAGE as described (5) and identified by MALDI-TOF analyses of tryptic peptides, which were formed by in gel digestion (44).
Enzyme Assay-Lipoxygenase activity was monitored by UV spectroscopy (at 235 and 237 nm; 10-mm path length in cuvettes of 0.15, 0.5, or 2 ml) in 0.1 M sodium borate buffer (pH 9.0), usually with 50 -100 M fatty acid substrates and 2-15% enzyme solution (by volume). Products were analyzed after extractive isolation (SepPak/C 18 or CH 2 Cl 2 ) and in some experiments reduced with NaBH 4 , NaB 2 H 4 , or 1-10 g of TPP (from 10 mg TPP/ml cyclohexane) before analysis. The transformation of 17:3n-3 and 19:3n-3 were compared with 18:3n-3 using the same concentrations of the substrates and the same amount of enzymes (Mn-LO and the G316A mutant as judged from their lipoxygenase activities with 18:3n-3 as a substrate).
The hydroperoxide isomerase activity was monitored by following the conversion of (13R)-HPOTrE (1-100 M) to 13-KOTrE by UV analysis at 280 nm in duplicate or triplicate, and the rate was determined from the linear part. Apparent K m was estimated by Michaelis-Menten kinetics. Conversion of (S)-and (R)-hydroperoxides to keto compounds was assayed by UV spectroscopy (280 nm). Hydroperoxide isomerase activity was also monitored by the decline in UV absorbance at 237 nm. The lipoxygenase inhibitors were dissolved in ethanol and preincubated for 5 min with the enzyme before the reaction started by the addition of substrate.
Oxygen-18 Experiment-The atmosphere over Mn-LO G316A (0.04 mg) in 6 ml of 0.1 M NaBO 3 buffer was repeatedly evacuated with an oil pump and flushed with nitrogen. Oxygen-18 (100 ml) was introduced from a sealed ampule under reduced pressure. 18:3n-6 was added to 100 M (in 0.1 ml of 0.1 M NaBO 3 saturated with N 2 ), and the reaction was terminated after 60 min with 2 ml of ethanol, diluted with 12 ml of water, and immediately extracted (SepPak/C 18 ).
Anaerobic Incubation-First, (13R)-HPODE and (13R)-HPOTrE were generated in situ. Recombinant Mn-LO (50 g) in 5 ml of 0.1 M NaBO 3 buffer, pH 9.0, was incubated under normal atmosphere with 100 M 18:2n-6 or 18:3n-3 until complete conversion of substrate had occurred (UV analysis). The vessel was then sealed, and the atmosphere was repeatedly evacuated with an oil pump and flushed with argon. Under anaerobic conditions, we added 50 g of Mn-LO (in 0.5 ml of 0.1 M NaBO 3 buffer, pH 9.0) and 18:2n-6 or 18:3n-3 to a final concentration of 100 M. The reaction proceeded for 1.5 h (21°C). It was terminated by the addition of methanol and followed by extractive isolation (14).
LC-MS Analysis-RP-HPLC with electrospray ionization was performed with an ion trap mass spectrometer (LCQ; Thermo) as described (46). The heated capillary was set to 215°C. In order to obtain ionization in the electrospray process, the SP-HPLC effluent (0.3 ml/min) was mixed with isopropyl alcohol/water (6/4; 0.2 ml/min) in a T junction and introduced to the mass spectrometer. An LTQ ion trap (Thermo) was used with a photodiode array detector (Surveyor, Thermo) in the later part of this study.
Spectroscopy-Light absorbance was measured with a dual beam spectrophotometer (Shimadzu UV-2101PC). The cis-trans conjugated hydro(pero)xy fatty acids were assumed to have an extinction coefficient of 25,000 cm Ϫ1 M Ϫ1 (5).
The kinetic UV trace (237 nm) showed that Mn-LO G316A also converted (13R)-HPOTrE to products with less UV absorption at this wavelength (Fig. 1A). To study this phenomenon, we compared the catalytic properties of the G316A mutant with recombinant Mn-LO, using 100 M 18:3n-3 as a substrate and similar amounts of enzyme, as judged from the initial rate of linear biosynthesis of cis-trans conjugated products. The native enzyme oxidized the substrate to completion after a kinetic time lag. After a similar kinetic time lag, Mn-LO G316A increased (13R)-HPOTrE formation linearly (maximal rate of 0.46 absorbance units/min at 100 M 18:3n-3), and it leveled off after consumption of ϳ75% of the substrate (after 5 min), and the amount of (13R)-HPOTrE then declined at a rate of 0.08 absorbance units/min. UV analysis showed that the decrease in absorbance at 237 nm was accompanied by an increase in absorbance at ϳ282 nm with an isosbestic point at 252 nm ( Fig. 1, inset). As described below, this was due to hydroperoxide isomerase activity of G316A.
Comparison with Mn-LO-Using 0.09 -0.7 M enzyme, it was possible to demonstrate that the native enzyme also possessed low but detectable hydroperoxide isomerase activity. A comparison of the hydroperoxide isomerase activities of Mn-LO and the G316A mutant with 18:3n-3 as a substrate is shown in Fig. 1B by following the decline in (13R)-HPOTrE. The decline in UV absorption was at least 7-fold faster with G316A. The transformation of (13R)-HPODE by Mn-LO was negligible in comparison with (13R)-HPOTrE (Fig. 1C). The n-3 double bond thus appeared to be essential for the hydroperoxide isomerase activity. As a percentage of the maximal lipoxygenase activity, the hydroperoxide isomerase activities of Mn-LO and the G316A mutant were ϳ1% and 7-8%, respectively. 17:3n-3 and 19:3n-3-Mn-LO seemed to oxygenate 50 M 17:3n-3 and 18:3n-3 with the same maximal rates ( Fig. 2A). The kinetic traces were also similar, but the products differed. 17:3n-3 was converted by Mn-LO mainly to 12-HPHTrE (oxidation at n-6), and only traces of 10-HPHTrE (oxidation of n-8; 1-2%) were detected during the linear part of the lipoxygenation curve (MS/MS analysis). The kinetic traces suggested that the relative rate of the hydroperoxide isomerase activity was slightly reduced with 17:3n-3 as a substrate in comparison with 18:3n-3 ( Fig. 2A).
19:3n-3 was a relatively poor substrate in comparison with 18:3n-3, but its kinetic trace showed several distinct features (Fig.  2C). A short kinetic time lag was followed by a relatively rapid and linear increase in UV absorbance at 237 mn (for 1-2 min), followed by a slower increase (for 2 min) and then a steady decline in UV absorbance (at the same rate as the decay of (13R)-HPOTrE). As a percentage of the maximal lipoxygenase activity, the hydroperoxide isomerase activity of Mn-LO with 19:3n-3 as a substrate was ϳ20%. The G316A mutant yielded a different kinetic trace with a monophasic linear increase in UV absorbance. This was followed first by a rapid decline (in comparison with 18:3n-3) and then a slow decline (Fig. 2D).
LC-MS/MS analysis showed that Mn-LO first transformed 19:3n-3 by oxidation at the n-8 carbon (to 12-HPNTrE) with less oxidation at the n-6 carbon (to 14-HPNTrE). In the initial phase (2 min), the relative amounts were 12-HPNTrE Ͼ 14-HPNTrE Ͼ 14-KNTrE (Fig. 3), but 2 min later, 14-KNTrE had accumulated as the main product with approximately equal amounts of 12-and 14-HPNTrE (data not shown). We expected G316A to favor oxidation at the n-8 carbon relative to the n-6 carbon, but LC-MS/MS analysis showed that 12-HPNTrE was less abundant than 14-HPNTrE during the linear phase.
Effect of Lipoxygenase Inhibitors-ETYA and BW A4C inhibit Mn-LO (6). We found that ETYA (100 M), a suicide-type inhibitor (47), reduced the rate of conversion of (13R)-HPOTrE to 13-KOTrE by Mn-LO G316A by 75% (Fig. 4A). BW A4C (100 M) is a metal-reducing lipoxygenase inhibitor and augmented the initial transformation rate of (13R)-HPOTrE to 13-KOTrE severalfold (Fig. 4A), but the drug then slowed down and blocked the reaction after a few min (cf. Ref. 48). This inhibitory effect was less pronounced when smaller amounts of enzyme were used.
The stimulatory effect of BW A4C on the hydroperoxide isomerase activity was concentration-dependent, as illustrated in Fig. 4B, with a severalfold increase in the initial rate of transformation of (13R)-HPOTrE to 13-KOTrE at 30 -100 M BW A4C, but the stimulatory effect declined at 300 M BW A4C. For comparison, 100 M   BW A4C only inhibited the lipoxygenase reaction by ϳ40% in these experiments.
Nordihydroguaiaretic acid (100 M) did not inhibit the lipoxygenase activity of the G316A mutant (or Mn-LO), and the drug had no effect on the initial rate of transformation of (13R)-HPOTrE, but it appeared to reduce the conversion of (13R)-HPOTrE after 7-8 min in analogy with this late effect of BW A4C (data not shown).
Product Identification-The products formed from 18:3n-3 by Mn-LO G316A were studied by LC-MS/MS (Fig. 5). RP-HPLC showed that Mn-LO G316A formed 11-HPOTrE and 13-HPOTrE in the same ratio as recombinant Mn-LO during the linear lipoxygenation phase (with a modest increase in 9-HOTrE as discussed above; data not shown). With time, 13-KOTrE and polar products accumulated. Reduction of the products with NaB 2 H 4 yielded 13-HOTrE with significant deuterium incorporation at C-13, whereas the deuterium incorporation at C-11 of 11-HOTrE was lower, indicating transformation of (11S)-HPOTrE to (13R)-HPOTrE rather than to 11-KOTrE. In addition to 13-KOTrE, 11-HOTrE (peak 4) and 13-HOTrE (peak 6, containing 7-8% 9-HOTrE) were detected (Fig. 5A). MS/MS analysis (m/z 309 3 full scan) of the polar products suggested that epoxyalcohols were formed, but attempts to resolve them by RP-HPLC yielded poor resolution. We therefore studied the products by LC-MS/MS in the straight phase mode.
The products formed by Mn-LO G316A after incubation with 18:3n-3 for 50 min were separated by SP-HPLC and analyzed by MS/MS as shown in Fig. 5, B and C. 13-KOTrE eluted as the least polar product. To simplify the analysis, hydroperoxides were reduced to alcohols with TPP and separated by SP-HPLC (Fig. 5B). At this time point, 13-HOTrE, 11-HODE, and 9-HODE eluted as major peaks along with small amounts of 12-HOTrE and 16-HOTrE. The MS/MS spectrum of 16-HOTrE showed a characteristic signal at m/z 233 due to loss of 58 (OCH-CH 2 -CH 3 ), and the MS/MS spectrum of 12-HOTrE showed a characteristic signal at m/z 211 ( Ϫ OOC-(CH 2 ) 7 -CHϭCH-CH 2 -HCO). (13R)-HPOTrE had apparently been almost completely converted to other products by Mn-LO G316A, and it was now present in smaller amounts than both 11-HOTrE and 9-HOTrE.
The most polar products were separated as shown in Fig. 5C. Based on systematic analysis of hematin-catalyzed isomerization of fatty acid hydroperoxides to epoxyalcohols discussed below and on authentic standards, the two major products were identified as erythro-and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acid (peaks 1 and 2), which were formed in a ratio of 2:3. In addition to these two trans-epoxides, the corresponding erythro and threo isomers of the corresponding cis-epoxide also appeared to be present as minor products   Effect of Oxygen Tension-We also investigated the effect of saturated oxygen concentration (1.1 mM O 2 ) on the hydroperoxide isomerase activity. In oxygen-saturated buffer, the maximal linear rate of biosynthesis of (13R)-HPOTrE was increased, but it leveled off after a few min. The UV absorption at 237 nm then declined at a rate 25% lower than in a parallel experiment with Mn-LO G316A under normal oxygen tension (0.22 mM O 2 ).

DISCUSSION
We report that site-directed mutagenesis of a single amino acid, G316A, in the active site of Mn-LO augmented its hydroperoxide isomerase activity severalfold and slightly shifted the position of oxygenation from the n-6 toward the n-8 and n-10 carbons. Mn-LO and its mutant catalyzed four reactions: hydrogen abstraction, oxygen insertion, migration of hydroperoxides at the n-8 carbon to the n-6 carbon (peroxide rearrangement), and transformation of n-6 hydroperoxides to epoxyalcohols and keto compounds. A discussion of these reactions would benefit from a hypothetical model where the substrate fatty acids enter the hydrophobic catalytic cavity with their methyl ends and with their carboxyl groups anchored, by ionic interaction, to a charge residue at a fixed position (28,42). The n-8 carbon should be close to the catalytic metal to allow lipoxygenation.
The first and rate-limiting step in Mn-LO catalysis is abstraction of the pro-S hydrogen at the n-8 carbon by the catalytic base, Mn 3ϩ OH (2). Mn-LO oxidized 17:3n-3 and 18:3n-3 at similar rates, whereas 19:3n-3 was oxidized slowly. The distance from the catalytic metal to the pro-S hydrogen at the n-8 carbon appeared to be essential for hydrogen abstraction and might contribute to the low turnover of 19:3n-3. The -electron interaction of the n-3 double bonds with aromatic amino acids probably orients these substrates with their n-8 carbons closer to the catalytic base, since both 18:2n-6 and 18:3n-6 are oxidized less efficiently than 18:3n-3 (6). The G316A mutant reduced the rate of oxygenation of 17:3n-3 in comparison with 18:3n-3, suggesting that the distance of n-8 carbon of 17:3n-3 to the catalytic base was increased. Whether the G316A mutation affected the absolute rate of lipoxygenation in comparison with Mn-LO will await further studies.
In the next step, the carbon-centered radical at the bis-allylic n-8 carbon reacts with molecular oxygen. The spin density is largest at the n-8 carbon (52), but oxygen insertion is controlled by steric factors. 17:3n-3 is oxidized by Mn-LO almost exclusively at the n-6 carbon (to 12-HPHTrE), whereas chain elongations to 18:3n-3 shifted oxygenation to 70 -80% at the n-6 carbon (to (13R)-HPOTrE) and 20 -30% at the n-8 carbon (to (11S)-HPOTrE) and chain elongations to 19:3n-3 shifted oxygenation to 20 -30% at the n-6 carbon (to (14R)-HPNTrE) and 70 -80% at the n-8 carbon (to (12S)-HPNTrE). Based on the model discussed above, these results suggest that the oxygenation channel only Mn-LOX G316A positioned some fatty acids so that the oxygen channel had access also to the n-8 and the n-10 carbons. The oxygenation of 17:3n-3 shifted from almost exclusively at the n-6 carbon toward the n-8 (7%) and n-10 (11%) carbons. The oxidation of 18:3n-3 by Mn-LO G316A was also shifted toward n-10 carbon with an increased formation of (9S)-HPOTrE. These results are in agreement with the shift in oxygenation of arachidonic acid from C-12 to C-8 by the Gly to Ala mutant of human (12R)-LOX but differ from the shift in arachidonate oxygenation, from C-8 to C-12, by the Gly to Ala mutant of coral (8R)-LOX (37,38).
The relative formation of hydroperoxides at the n-6 and n-8 carbons can also be affected by migration of hydroperoxides at n-8 to n-6 (cf. Fig.  9), since the n-6 hydroperoxides accumulate as the end products (2). During the linear phase, Mn-LO thus transforms 18:3n-3 to (13R)-HPOTrE and (11S)-HPOTrE in a ratio of ϳ3-4:1. When the concentration of the substrate declines, (11S)-HPOTrE is converted to (13R)-HPOTrE in a less efficient process than the lipoxygenation. The kinetic analysis of 19:3n-3 suggested a similar mechanism. Rapid transformation of 19:3n-3 by Mn-LO to (12S)-HPNTrE and (14R)-HPNTrE with consumption of the substrate was followed by migration of the hydroperoxide group from n-8 to n-6 to yield the end product, (14R)-HPNTrE, at a lower rate.
Analysis of the reaction mechanism showed that both hydroperoxide oxygen atoms were retained in the epoxyalcohols. The reaction mechanism is described schematically in Fig. 10. The reaction was inhibited by ETYA, modestly reduced by increased oxygen tension, and stimulated by a metal-reducing lipoxygenase inhibitor (BW A4C). Importantly, the hydroperoxide isomerase activity was not dependent on metal-reducing agents and thus different from the pseudoperoxidase activity of sLO-1 and 5-LOX observed in the presence of a reducing agent (19,20).
The hydroperoxide isomerase activity of Mn-LO probably requires that the hydroperoxide group be positioned in close vicinity to the reduced catalytic metal. The interaction of the n-3 double bond with -electrons of aromatic amino acids appeared to be important also in this context, since (13R)-HPODE was a very poor substrate of the hydroperoxide isomerase activity of Mn-LO (and Mn-LO G316A). The chain length also influenced the reaction. Mn-LO exhibited approximately the same and relatively low hydroperoxide isomerase activity toward (14R)-HPNTrE and (13R)-HPOTrE, whereas (12R)-HPHTrE appeared to be transformed less efficiently than (13R)-HPOTrE.
It is of interest to compare the reaction mechanism of Mn-LO G316A with eLOX3, which has been characterized in detail (11). Both enzymes catalyze retention of the original hydroperoxide oxygens in the epoxyalcohols in hydroperoxide isomerase reactions but form different  The catalytic metal is oxidized by hydroperoxides (LOOH) to Mn 3ϩ OH, and an alkoxyl radical is formed (LO ⅐ ). In the next step, the alkoxyl radical is transformed by the enzyme to epoxyalcohols or to a keto fatty acid and water with retention of both hydroperoxyl oxygens in the epoxyalcohol, and the catalytic metal is reduced to Mn 2ϩ . BW A4C presumably stimulates the reaction by reducing Mn 3ϩ to Mn 2ϩ , whereas ETYA inhibited the reaction, as indicated. epoxyalcohols. eLOX3 transformed (12R)-HPETE to (8R)-hydroxy-(11R,12R)-epoxyeicosatrienoic acid and 12-KETE and transformed (15S)-HPETE to threo-13-hydroxy-(14S,15S)-epoxyeicosatrienoic acid and 15-KETE and (12S)-HPETE to at least two epoxyalcohols and to 12-KETE (11). eLOX3 was stimulated ϳ4-fold by nordihydroguaiaretic acid, which probably reduces Fe 3ϩ to Fe 2ϩ , whereas a hydroxamic acid derivative (BW A4C) stimulated the hydroperoxide isomerase activity of Mn-LO G316A ϳ5-fold, presumably by reducing Mn 3ϩ to Mn 2ϩ (20). BW A4C inhibited lipoxygenation by the same mechanism. In contrast to eLOX3, nordihydroguaiaretic acid did not stimulate the hydroperoxide isomerase activity of Mn-LO G316A, and the drug did not block its lipoxygenase activity.
In summary, the reduced catalytic metals initiate homolytic cleavage of the hydroperoxide oxygen-oxygen bond of the fatty acid and form an alkoxyl radical and Mn 3ϩ OH and Fe 3ϩ OH, respectively. The alkoxyl radical rearranges to a keto fatty acid and to a trans-epoxide with a carbon-centered radical, to which oxygen is rebound with formation of epoxyalcohols (or reduced to water with formation of keto fatty acids). In this step, the catalytic metal is reduced to Mn 2ϩ and Fe 2ϩ , and a new hydroperoxide isomerase cycle may begin, as outlined in Fig. 10.
What prevents some lipoxygenases from catalyzing efficient hydroperoxide isomerase reactions under normal oxygen tension in analogy with eLOX3 and Mn-LO G316A? The reason may be related to the redox potential of the catalytic metal and to steric factors. The redox potentials (e o , V) are as follows: Fe 3ϩ ϩ e Ϫ 3 Fe 2ϩ (ϩ0.77), and Mn 3ϩ ϩ e Ϫ 3 Mn 2ϩ (ϩ1.51). Mn 2ϩ is thus more difficult to oxidize than Fe 2ϩ . Brash and co-workers (11) proposed that the ferric redox state of eLOX3 was unfavorable. The stable ferrous form made eLOX3 incapable of performing abstraction of bis-allylic hydrogens of polyunsaturated fatty acids and lipoxygenation, but the ferrous form catalyzed the hydroperoxide isomerase activity. It seems unlikely that the Gly 3 Ala mutation of Mn-LO significantly changed the redox potential of the ligated manganese, but it may position the (R)-hydroperoxide groups closer to the catalytic metal.
The two bulky mutations, G316V and G316T, were catalytically inactive, as reported for the corresponding two mutations of (12R)-LOX (37)(38)(39). It was more surprising that G316S also was catalytically inactive. We confirmed that G316S did not possess hydroperoxide isomerase activity.
In conclusion, the Coffa and Brash determinant Ala/Gly for position and S/R stereospecificity of lipoxygenases is also relevant for Mn-LO. In addition, Ala/Gly may also be a determinant of hydroperoxide isomerase activity. eLOX3 has Ala in this position. The hydroperoxide isomerase reaction of eLOX3 is catalyzed by Fe 2ϩ , which redox-cycles in this process. Our results show that this selfsufficient catalytic cycle of eLOX3 also is present in Mn-LO and can be augmented by mutation of Gly-316 to Ala. This provides new insight into the reaction mechanism of hydroperoxide isomerases and properties of manganese-and iron-containing lipoxygenases.