Separation of enzymatic functions and variation of spin state of rice allene oxide synthase-1 by mutation of Phe-92 and Pro-430

Rice allene oxide synthase-1 mutants carrying F92L, P430A or F92L/P430A amino acid substitution mutations were constructed, recombinant mutant and wild type proteins were purified and their substrate preference, UV–vis spectra and heme iron spin state were characterized. The results show that the hydroperoxide lyase activities of F92L and F92L/P430A mutants prefer 13-hydroperoxy substrate to other hydroperoxydienoic acids or hydroperoxytrienoic acids. The Soret maximum was completely red-shifted in P430A and F92L/P430A mutants, but it was partially shifted in the F92L mutant. ESR spectral data showed that wild type, F92L and P430A mutants occupied high and low spin states, while the F92L/P430A mutant occupied only low spin state. The extent of the red shift of the Soret maximum increased as the population of low spin heme iron increased, suggesting that the spectral shift reflects the high to low transition of heme iron spin state in rice allene oxide synthase-1. Relative to wild type allene oxide synthase-1, the hydroperoxide lyase activities of F92L and F92L/P430A are less sensitive to inhibition by imidazole with (13S or 9S)-hydroperoxydienoic acid as substrate and more sensitive than wild type with (13S)-hydroperoxytrienoic acid as substrate. Our results suggest that hydroperoxydienoic acid is the preferred substrate for the hydroperoxide lyase activity and (13S)-hydroperoxytrienoic acid is the preferred substrate for allene oxide synthase activity of allene oxide synthase-1.     

Regioselective and stereospecific glucuronidation of trans- and cis-resveratrol in human

A novel member of the plant cytochrome P450 CYP74 family of fatty acid hydroperoxide metabolizing enzymes has been cloned from melon fruit (Cucumis melo). The cDNA is comprised of 1,446 nucleotides encoding a protein of 481 amino acids. The homology at the amino acid level to other members of the CYP74 family is 35-50%, the closest relatives being allene oxide synthases. The cDNA was expressed in Escherichia coli, and the corresponding protein was purified by affinity column chromatography. The native enzyme showed a main Soret band at 418 nm, indicative of a low spin ferric cytochrome P450, and a 447-nm peak appeared in the CO-difference spectrum. Using [U-14C]radiolabeled substrate, HPLC, UV, and GC-MS, the products of conversion of 9S-hydroperoxy-linoleic acid were identified as 9-oxo-nonanic acid and 3Z-nonenal. Kinetic analysis of this hydroperoxide lyase showed the highest rate of reaction with 9-hydroperoxy-linolenic acid followed by 9-hydroperoxy-linoleic acid and then the corresponding 13-hydroperoxides. Overall, the newly characterized cytochrome P450 enzyme is a fatty acid hydroperoxide lyase with a preference, but not absolute specificity for the 9-positional hydroperoxides of linoleic and linolenic acids.     

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.     

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.     

Identification of an allene oxide synthase (CYP74C) that leads to formation of alpha-ketols from 9-hydroperoxides of linoleic and linolenic acid in below-ground organs of potato

Allene oxide synthase (AOS) enzymes are members of the cytochrome P450 enzyme family, sub-family CYP74. Here we describe the isolation of three cDNAs encoding AOS from potato (StAOS1-3). Based on sequence comparisons, they represent members of either the CYP74A (StAOS1 and 2) or the CYP74C (StAOS3) sub-families. StAOS3 is distinguished from the other two AOS isoforms in potato by its high substrate specificity for 9-hydroperoxides of linoleic and linolenic acid, compared with 13-hydroperoxides, which are only poor substrates. The highest activity was shown with (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) as a substrate. This hydroperoxide was metabolized in vitro to alpha- and gamma-ketols as well as to the cyclopentenone compound 10-oxo-11-phytoenoic acid. They represent hydrolysis products of the initial StAOS3 product 9,10-epoxyoctadecadienoic acid, an unstable allene oxide. By RNA gel hybridization blot analysis, StAOS3 was shown to be expressed in sprouting eyes, stolons, tubers and roots, but not in leaves. StAOS3 protein was found in all organs tested, but mainly in stems, stolons, sprouting eyes and tubers. As in vivo reaction products, the alpha-ketols derived from 9-hydroperoxides of linoleic and linolenic acid were only found in roots, tubers and sprouting eyes. Immunolocalization showed that StAOS3 was associated with amyloplasts and leucoplasts.     

Molecular cloning of allene oxide cyclase. The enzyme establishing the stereochemistry of octadecanoids and jasmonates

Allene oxide cyclase (EC ) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5'-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13S enantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.     

Detection of unprecedented allene oxide synthase member of CYP74B subfamily: CYP74B33 of carrot (Daucus carota)

Enzymes of CYP74 family widespread in higher plants control the metabolism of fatty acid hydroperoxides to numerous bioactive oxylipins. Hydroperoxide lyases (HPLs, synonym: hemiacetal synthases) of CYP74B subfamily belong to the most common CYP74 enzymes. HPLs isomerize the hydroperoxides to the short-lived hemiacetals, which are spontaneously decomposed to aldehydes and aldoacids. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) possessed the 13-HPL activity. Present work reports the cloning of the expressed CYP74B33 gene of carrot (Daucus carota L.) and studies of catalytic properties of the recombinant CYP74B33 protein. In contrast to all CYP74B proteins studied yet, CYP74B33 behaved differently in few respects. Firstly, the preferred substrates of CYP74B33 are 9-hydroperoxides. Secondly and most importantly, CYP74B33 exhibits the 9-allene oxide synthase (AOS) activity. For example, the 9(S)-hydroperoxide of linoleic acid (9-HPOD) underwent the conversion to α-ketol via the short-lived allene oxide. Uncommonly, the 9-HPOD conversion affords a minority of cis-10-oxo-11-phytoenoic acid, which is also produced by CYP74C but not the CYP74A AOSs. The similar product patterns were observed upon the incubations of CYP74B33 with 9(S)-hydroperoxide of α-linolenic acid. The enzyme possessed a mixed HPL, AOS, and the epoxyalcohol synthase activity toward the 13-hydroperoxides, but the total activity was much lower than toward 9-hydroperoxides. Thus, the obtained results show that CYP74B33 is an unprecedented 9-AOS within the CYP74B subfamily.     

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.     

Biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid: identification of an allene oxide cyclase

Incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn (Zea mays L.) hydroperoxide dehydrase led to the formation of an unstable allene oxide derivative, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid. Further conversion of the allene oxide yielded two major products, i.e. alpha-ketol 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid, and 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA). 12-Oxo-PDA was formed from allene oxide by two different pathways, i.e. spontaneous chemical cyclization, leading to racemic 12-oxo-PDA, and enzyme-catalyzed cyclization, leading to optically pure 12-oxo-PDA. The allene oxide cyclase, a novel enzyme in the metabolism of oxygenated fatty acids, was partially characterized and found to be a soluble protein with an apparent molecular weight of about 45,000 that specifically catalyzed conversion of allene oxide into 9(S),13(S)-12-oxo-PDA.     

Geometrical Configuration of 12,13-Epoxyoctadeca-9,11-dienoic Acid,a Product of the Reaction Catalyzed by Flaxseed Allene Oxide Synthase (CYP74A)

The geometrical configuration of a short-living allene oxide reaction product that arises under the catalysis by flaxseed allene oxide synthase (CYP74A) was studied by NMR spectroscopy. The structure of (9Z, 11E)-12,13-epoxyoctadeca-9,11-dienoic acid was established for it from the results of the nuclear Overhauser effect measurements.     

Geometrical configuration of 12,13-epoxy-9,11-octadecadienoic acid, a product of the reaction catalyzed by flaxseed allene oxide synthase (CYP74A)

The geometrical configuration of a short-living allene oxide reaction product that arises under the catalysis by flaxseed allene oxide synthase (CYP74A) was studied by NMR spectroscopy. The structure of (9Z,11E)-12,13-epoxyoctadeca-9,11-dienoic acid was established for it from the results of the nuclear Overhauser effect. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Identification and functional analyses of two cDNAs that encode fatty acid 9-/13-hydroperoxide lyase (CYP74C) in rice

Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.     

Allene oxide cyclase: a new enzyme in plant lipid metabolism

The mechanism of the biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA) from 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid in preparations of corn (Zea mays L.) was studied. In the initial reaction the hydroperoxide was converted into an unstable allene oxide, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, by action of a particle-bound hydroperoxide dehydrase. A new enzyme, allene oxide cyclase, catalyzed subsequent cyclization of allene oxide into 9(S),13(S)-12-oxo-PDA. In addition, because of its chemical instability, the allene oxide underwent competing nonenzymatic reactions such as hydrolysis into alpha- and gamma-ketol derivatives as well as spontaneous cyclization into racemic 12-oxo-PDA. (+/-)-cis-12,13-Epoxy-9(Z)-octadecenoic acid and (+/-)-cis-12,13-epoxy-9(Z),15(Z)-octadecadienoic acid, in which the epoxy group was located in the same position as in the allene oxide substrate, served as potent inhibitors of corn allene oxide cyclase. On the other hand, the isomeric (+/-)-cis-9,10-epoxy-12(Z)-octadecenoic acid had little inhibitory effect. Allene oxide cyclase was present in the soluble fraction of corn homogenate and had a molecular weight of about 45,000 as judged by gel filtration. The enzyme activity was detected in several plant tissues, the highest levels being observed in potato tubers and in leaves of spinach and white cabbage.     

Determinants governing the CYP74 catalysis: conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the "I-helix central domain" ("oxygen binding domain") are the primary determinants of CYP74 type of catalysis.     

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3: STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES*

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at −15 °C. One matched previously described allene oxides in its UV spectrum (λ_max 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λ_max 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.