Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of ∼50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-²H]18:1n-9, [7R-²H]18:2n-6, and [8R-²H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.     

Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase,and 8,11-hydroperoxide isomerase of Aspergillus clavatus

Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity (∼ 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain (∼ 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10R-DOX (ppoC) of Aspergillusnidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8R-HPODE was then isomerized to 5S,8R-DiHODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with > 65% amino acid identity to 10R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE.     

Gene Deletion of 7,8-Linoleate Diol Synthase of the Rice Blast Fungus: STUDIES ON PATHOGENICITY, STEREOCHEMISTRY, AND OXYGENATION MECHANISMS*

Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Δ7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Δ7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Δmac1 sum1–99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.     

On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.)

Seeds of broad bean (Vicia faba L.) contain a hydroperoxide-dependent fatty acid epoxygenase. Hydrogen peroxide served as an effective oxygen donor in the epoxygenase reaction. Fifteen unsaturated fatty acids were incubated with V. faba epoxygenase in the presence of hydrogen peroxide and the epoxy fatty acids produced were identified. Examination of the substrate specificity of the epoxygenase using a series of monounsaturated fatty acids demonstrated that (Z)-fatty acids were rapidly epoxidized into the corresponding cis-epoxy acids, whereas (E)-fatty acids were converted into their trans-epoxides at a very slow rate. In the series of (Z)-monoenoic acids, the double bond position as well as the chain length influenced the rate of epoxidation. The best substrates were found to be palmitoleic, oleic, and myristoleic acids. Steric analysis showed that most of the epoxy acids produced from monounsaturated fatty acids as well as from linoleic and α-linolenic acids had mainly the (R),(S) configuration. Exceptions were C₁₈ acids having the epoxide group located at C-12/13, in which cases the (S),(R) enantiomers dominated. 13(S)-Hydroxy-9(Z),11(E)-octadecadienoic acid incubated with epoxygenase afforded the epoxy alcohol 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as the major product. Smaller amounts of the diastereomeric epoxy alcohol 9(R),10(S)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as well as the α,β-epoxy alcohol 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid were also obtained. The soluble fraction of homogenate of V. faba seeds contained an epoxide hydrolase activity that catalyzed the conversion of cis-9,10-epoxyoctadecanoic acid into threo-9,10-dihydroxyoctadecanoic acid.     

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C₁₆ and C₁₈ fatty acids at pH ∼7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.The fungus Aspergillus nidulans (teleomorph Emericella nidulans) is a homothallic ascomycete that has a defined sexual and asexual developmental cycle. Therefore, it serves as a model system for the understanding of fungal development (1). Oxidized unsaturated fatty acids, so-called oxylipins, derived from endogenous fatty acids were found to influence the development of the asexual conidiophores and sexual cleistothecia (2–6). Moreover, they seem to regulate the secondary metabolism of the fungus (7). These substances were collectively named psi factors and are primarily a mixture of hydroxylated oleic (18:1^Δ9Z; x:y^Δz denotes a fatty acid with x carbons and y double bonds in position z counting from the carboxyl end), linoleic (18:2^Δ9Z,12Z), and α-linolenic (18:3^Δ9Z,12Z,15Z) acids. They are termed psiβ, psiα, and psiγ, respectively. Psi factors can be further classified by the number and positioning of hydroxy groups on the fatty acid backbone: psiB (OH at C-8, e.g. (8R)-HODE),² psiA (OH at C-5 and C-8, e.g. (5S,8R)-DiHODE), and psiC (OH at C-8 and the δ-lactone ring) (8, 9).The psi factor (8R)-HODE was first discovered in the fungus Laetisaria arvalis (10, 11); it was later also found in Gaeumannomyces graminis (12, 13), where the first enzyme, which is responsible for production of (8R)-HPODE, 7,8-LDS, was detected (13). This heme-containing enzyme is bifunctional because it oxidizes 18:2^Δ9Z,12Z in a first reaction step to (8R)-HPODE and subsequently isomerizes this intermediate compound to (7S,8S)-DiHODE (13–15).After the genome of A. nidulans was available, Keller and co-workers (6, 16, 17) found three genes that share a high homology with the sequence of 7,8-LDS, namely ppoA, ppoB, and ppoC. They showed that the deletion of these genes had a significant effect (i) on the developmental ratio between the asexual conidiospores and sexual ascospores; (ii) on the production of psi factors; and (iii) on the production of secondary metabolites, the mycotoxins (6, 7, 16, 17). Furthermore, the encoded proteins showed remarkable sequence homology to both mammalian PGHS isoforms, enzymes that are responsible for the synthesis of prostaglandins (18). Using the NCBI conserved domain search analysis tool, it turned out that ppoA amino acid residues 210–580 contain a domain similar to mammalian heme peroxidases, whereas residues 650–1050 contain a CYPX domain, similar to P450 heme thiolate enzymes (16). However, for 7,8-LDS from G. graminis, only the mammalian heme peroxidase domain is predicted. The identity of conserved catalytic domains between Ppo enzymes and mammalian PGHS ranges from 25 to 29% for PGHS-2 and from 25 to 26% for PGHS-1 (19). PpoA and 7,8-LDS show 42% amino acid identity.Oliw and co-workers (20) observed that incubation of homogenates of mycelia of A. nidulans with 18:2^Δ9Z,12Z converted the fatty acid to (8R)-HODE and (5S,8R)-DiHODE as the major products. (8R)-HPODE, (10R)-HODE, and (10R)-HPODE were detected as minor products. Incubation of mycelia of Aspergillus fumigatus with deuterium-labeled 18:2^Δ9Z,12Z revealed that the synthesis of (8R)-HPODE is accomplished via pro-S-hydrogen abstraction at C-8 and antarafacial dioxygen insertion. (5S,8R)-DiHODE is generated via an additional pro-S-hydrogen abstraction at C-5 of the substrate (20, 21).Additional studies with fungal knock-out strains led to the hypothesis that PpoA may be responsible for the synthesis of (8R)-hydroperoxides, which are partially reduced to (8R)-hydroxides (20). It was suggested that, analogous with 7,8-LDS, (8R)-hydroperoxides are then converted to 5,8-dihydroxides by PpoA. Furthermore, it was concluded that ppoC may code for linoleate (10R)-DOX (20). Analysis of Ppo enzymes from A. nidulans in studies published so far has been performed either by using knock-out mutants to demonstrate the absence of a subset of psi factors or by using crude mycelial extracts; both experimental setups have the disadvantage of observing multiple enzymatic reactions in parallel.To characterize the biochemical properties of PpoA in more detail, we cloned and expressed recombinant PpoA in Escherichia coli. After purification of the enzyme by up to 62-fold, biochemical characterization was performed. The studies revealed mechanistic as well as structural similarities to and differences from 7,8-LDS from G. graminis. Both enzymes were found to be homotetrameric ferric heme proteins that catalyze the synthesis of (8R)-HPODE. Whereas G. graminis 7,8-LDS converts the intermediate formed to (7S,8S)-DiHODE, PpoA produces 5,8-DiHODE.Using site-directed mutagenesis, we provide evidence that there are striking differences between both enzymes regarding the catalytic reaction cycle. Thus, we found that PpoA uses different domains to catalyze the two reaction steps. We suggest that the DOX reaction, yielding 8-HPODE, takes place in the N-terminal heme peroxidase domain. The isomerization of this intermediate product to the end product, 5,8-DiHODE, is accomplished, however, independently by the C-terminal P450 heme thiolate domain in an 8-hydroperoxide isomerase reaction.In addition, we are able to provide evidence that, during the catalysis, PpoA generates a carbon-centered radical presumably at C-8, like G. graminis 7,8-LDS. Furthermore, we determined the kinetic parameters for the first reaction step.     

Secretion of two novel enzymes,manganese 9S-lipoxygenase and epoxy alcohol synthase,by the rice pathogen Magnaporthe salvinii

The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion.     

Molecular characterization of Penicillium oxalicum 6R,8R-linoleate diol synthase with new regiospecificity

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn⁸⁸⁶, Arg⁷⁰⁷, and Arg⁹³⁴, are catalytic importance and belong to the catalytic groove. Phe⁷⁹⁴ and Gln⁸⁸⁹ were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C₁₈ fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.     

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-²H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the ²H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser³⁴⁸ in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser³⁴⁸, binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.     

Ring A-stereoisomers of 1-hydroxyvitamin D3 and their relative binding affinities for the intestinal 1α,25-dihydroxyvitamin D3 receptor protein

A set of eight 1-hydroxyvitamin D₃ compounds comprising the four possible (5Z)-1,3-diol stereoisomers and the corresponding (5E)-double bond isomers, has been prepared in order to assess the effect of 1,3-diol stereochemistry and 5,6-double bond geometry on binding affinity for the intestinal 1,25-(OH)₂D₃-receptor protein. The compounds were synthesized from either vitamin D₃ or 3-epivitamin D₃ via 3,5-cyclovitamin D intermediates. Competitive receptor binding assays establish that all changes from the natural ring A-configuration (1S, 3R, 5Z) lead to decreased binding affinity, and confirm the importance of the 1-hydroxy function since the conversion of stereochemistry at that center from 1α(S) to 1β(R) has the most pronounced effect on binding affinity (attenuation by more than three orders of magnitude). Other modifications (i.e., conversion at C-3, or cis to trans isomerization of the 5,6-double bond) decrease binding affinity by more moderate (ca. 10-fold) but cumulative factors.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)- and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY*S?

Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; ∼90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; ∼10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms ∼98% 8R-HPODE and ∼2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.Linoleate diol synthases (LDS)² and linoleate 10R-DOX are fungal fatty acid dioxygenases of the myeloperoxidase gene family (1-3). LDS have dual enzyme activities and transform 18:2n-6 sequentially to 8R-HPODE in an 8R-dioxygenase reaction and to 5,8-, 7,8-, or 8,11-DiHODE in hydroperoxide isomerase reactions. These oxylipins affect sporulation, development, and pathogenicity of Aspergilli (4-6). Fatty acid dioxygenases of the myeloperoxidase gene family also occur in vertebrates, plants, and algae (7-9). The most thoroughly investigated vertebrate enzymes are ovine PGHS-1 and mouse PGHS-2 with known crystal structures (10-12). PGHS transforms 20:4n-6 to PGG₂ in a cyclooxygenase and PGG₂ to PGH₂ in a peroxidase reaction. Aspirin and other nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase reaction. This is of paramount medical importance (13, 14), and PGHS-1 and -2 are commonly known as COX-1 and -2 (15). α-DOX occur in plants and algae, and biosynthesis of α-DOX in plants is elicited by pathogens (7). α-DOX oxidizes fatty acids to unstable (2R)-hydroperoxides, which readily break down nonenzymatically to fatty acid aldehydes and CO₂ (7).LDS, 10R-DOX, PGHS, and α-DOX oxygenate fatty acids to different products, but their oxygenation mechanisms have mechanistic similarities. Sequence alignment shows that many critical amino acid residues for the cyclooxygenase reaction are conserved in LDS, 10R-DOX, and α-DOX. These include the proximal histidine heme ligand, the distal histidine, and the catalytic important tyrosine (Tyr-385) of PGHS-1. The latter is oxidized to a tyrosyl radical, which initiates the cyclooxygenase reaction by abstraction of the pro-S hydrogen at C-13 of 20:4n-6 (16). In analogy, LDS and 10R-DOX catalyze stereospecific abstraction of the pro-S hydrogen at C-8 of 18:2n-6 (3), whereas α-DOX abstracts the pro-R hydrogen at C-2 of fatty acids (17). Site-directed mutagenesis of the conserved tyrosine homologues of Tyr-385 and proximal heme ligands abolishes the dioxygenase activities of 7,8-LDS and α-DOX (17, 18). The orientation of the substrate at the dioxygenation site differs. The carboxyl groups of fatty acids are positioned in a hydrophobic grove close to the tyrosine residue of α-DOX (19). In contrast, the ω ends of eicosanoic fatty acids are buried deep inside the cyclooxygenase channel so that C-13 lies in the vicinity of Tyr-385 (20). Several observations suggest that 18:2n-6 may also be positioned with its ω end embedded in the interior of 7,8-LDS of Gaeumannomyces graminis (18).7,8-LDS of G. graminis and Magnaporthe grisea and 5,8-LDS of Aspergillus nidulans have been sequenced (5, 8, 21). Gene targeting revealed the catalytic properties of 5,8-LDS, 8,11-LDS, and 10R-DOX in Aspergillus fumigatus and A. nidulans (3). Homologous genes can be found in other Aspergilli spp. Alignment of the two 7,8-LDS amino acid sequences with 5,8-LDS, 8,11-LDS, and 10R-DOX sequences of five Aspergilli revealed several conserved regions with single amino acid differences between the enzymes with 8R-DOX and 10R-DOX activities, as illustrated by the selected sequences in Fig. 1. Leu-306, Leu-384, and Val-388 of 10R-DOX are replaced in 5,8- and 7,8-LDS by valine, valine, and leucine residues, respectively. Whether these amino acids are important for the oxygenation mechanism is unknown, and this is one topic of the present investigation. The predicted secondary structure of 10R-DOX suggests that Leu-384 of 10R-DOX can be present in an α-helix with Val-388 close to its border. This α-helix is homologous to helix 6 of PGHS-1, which contains Val-349 and Ser-353 at the homologous positions of Leu-384 and Val-388 (Fig. 1).Open in a separate windowFIGURE 1.Alignments of partial amino acid sequences of five heme containing fatty acid dioxgenases and a comparison of the predicted secondary structure of 10R-DOX with ovine PGHS-1. A, top, amino acids residues at the presumed peroxidase and hydroperoxide isomerase sites. The last two residues, His and Asn, are conserved in all myeloperoxidases (1). Middle and bottom, amino acid residues of the presumed dioxygenation sites are shown. Conserved residues in all sequences are in boldface, and mutated residues of 10R-DOX and/or 7,8-LDS are marked by an asterisk. B, alignment of partial amino acid sequences of 10R-DOX with ovine PGHS-1, and a secondary structure prediction of the 10R-DOX sequence. The secondary structure of 10R-DOX was predicted by PSIPRED (43) and the secondary structure of ovine PGHS-1 from its crystal structure (Protein Data Bank code 1diy; cf. Ref 19). In short, our first strategy for site-directed mutagenesis was to switch hydrophobic residues between the enzymes with 10R- and 8R-DOX activities and to assess the effects on the DOX and hydroperoxide isomerase activities (10R-DOX/7,8-LDS: Leu-306/Val-256, Leu-384/Val-330, Val-388/Leu-334, and Ala-426/Ile-375) and to switch one hydrophobic/charged residue (Ala-435/Glu-384). Only catalytically active pairs would provide clear information on their importance for the position of dioxygenation (e.g. L384V of 10R-DOX and V330L of 7,8-LDS, both of which were active). Unfortunately, replacements of 7,8-LDS often led to inactivation or very low activity (e.g. V330A, V330M, I375A, E384A). Our second strategy was to study replacements in two homologous positions of ovine PGHS-1 (Val-349 and Ser-353) with smaller and larger hydrophobic residues, i.e. at Leu-384 and Val-388 of 10R-DOX. Abbreviations used are as follows: oCOX-1, ovine cyclooxygenase-1; Af, A. fumigatus; Gg, G. graminis. The GenBank™ protein sequences were derived from {"type":"entrez-protein","attrs":{"text":"P05979","term_id":"754286265","term_text":"P05979"}}P05979, {"type":"entrez-protein","attrs":{"text":"EAL89712","term_id":"66849384","term_text":"EAL89712"}}EAL89712, {"type":"entrez-protein","attrs":{"text":"AAD49559","term_id":"258406875","term_text":"AAD49559"}}AAD49559, {"type":"entrez-protein","attrs":{"text":"EAL84400","term_id":"129555261","term_text":"EAL84400"}}EAL84400, and {"type":"entrez-protein","attrs":{"text":"ACL14177","term_id":"219382647","term_text":"ACL14177"}}ACL14177. The amino acid sequences were aligned with the ClustalW algorithm (DNAStar).The overall three-dimensional structures of myeloperoxidases are conserved. It is therefore conceivable that important residues for substrate binding in the cyclooxygenase channel of PGHS could be conserved in LDS and 10R-DOX. The three-dimensional structure of ovine PGHS-1 shows that Val-349 and Ser-353 are close to C-3 and C-4 of 20:4n-6, and residues in these positions can alter both position and stereospecificity of oxygenation (22-24). Replacement of Val-349 of PGHS-1 with alanine increased the biosynthesis of 11R-HETE, whereas V349L decreased the generation of 11R-H(P)ETE and increased formation of 15(R/S)-H(P)ETE (23, 25). V349I formed PGG₂ with 15R configuration (22, 24). Replacement of Ser-353 with threonine reduced cyclooxygenase and peroxidase activities by over 50% and increased the biosynthesis of 11R-HPETE and 15S-HPETE 4-5 times (23).There is little information on the hydroperoxide isomerase and peroxidase sites of LDS (18, 26), but the latter could be structurally related to the peroxidase site of PGHS. PGG₂ and presumably 8R-HPODE bind to the distal side of the heme group, which can be delineated by hydrophobic amino acid residues (27). Val-291 is one of these residues, which form a dome over the distal heme side of COX-1. The V291A mutant retained cyclooxygenase and peroxidase activities (27). 5,8- and 7,8-LDS also have valine residues in the homologous position, whereas 8,11-LDS and 10R-DOX have leucine residues (Fig. 1). Whether these hydrophobic residues are important for the peroxidase activities is unknown.In this study we decided to compare the two catalytic sites of 10R-DOX of A. fumigatus and 7,8-LDS (EC 1.13.11.44) of G. graminis (18). Our first aim was to find a robust expression system for 10R-DOX of A. fumigatus. The second objective was to determine whether C₁₆ and C₂₀ fatty acid substrates enter the oxygenation site of 10R-DOX “head” or “tail” first. Unexpectedly, we found that 10R-DOX oxygenated 20:4n-6 by hydrogen abstraction at both C-13 and C-10 with formation of two nonconjugated and four cis-trans-conjugated HPETEs. Our third objective was to investigate the structural differences between 10R-DOX and 7,8-LDS of G. graminis, which could explain that oxygenation of 18:2n-6 mainly occurred at C-10 and at C-8, respectively. The strategy for site-directed mutagenesis of 10R-DOX and 7,8-LDS is outlined in the legend to Fig. 1; an alignment of the amino acid sequences of 10R-DOX and 7,8-LDS is found in supplemental material.     

Cineole biodegradation: Molecular cloning,expression and characterisation of (1R)-6β-hydroxycineole dehydrogenase from Citrobacter braakii

The first steps in the biodegradation of 1,8-cineole involve the introduction of an alcohol and its subsequent oxidation to a ketone. In Citrobacter braakii, cytochrome P450_cin has previously been demonstrated to perform the first oxidation to produce (1R)-6β-hydroxycineole. In this study, we have cloned cinD from C. braakii and expressed the gene product, which displays significant homology to a number of short-chain alcohol dehydrogenases. It was demonstrated that the gene product of cinD exhibits (1R)-6β-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1R)-6β-hydroxycineole was converted to (1R)-6-ketocineole. The (1R)-6β-hydroxycineole dehydrogenase exhibited a strict requirement for NAD(H), with no reaction observed in the presence of NADP(H). The enzyme also catalyses the reverse reaction, reducing (1R)-6-ketocineole to (1R)-6β-hydroxycineole. During this study the N-terminal His-tag used to assist protein purification was found to interfere with NAD(H) binding and lower enzyme activity. This could be recovered by the addition of Ni²⁺ ions or proteolytic removal of the His-tag.     

Oxidation of Two Hydroxylated Ochratoxin A Metabolites by Alcohol Dehydrogenase

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. K_m and turnover number for 10-hydroxyochratoxin A were 110 μM and 0.1 s⁻¹, respectively.     

Novel insights into cyclooxygenases,linoleate diol synthases,and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O₂. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n ? 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n ? 6 and n ? 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C₂₀ and C₁₈ fatty acids in the following order of rates: 20:2n ? 6 > 20:1n ? 6 > 20:3n ? 9 > 20:1n ? 9 and 18:3n ? 3 ≥ 18:2n ? 6 > 18:1n ? 6. 18:2n ? 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n ? 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n ? 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-²H]18:2n ? 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n ? 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-²H₂]- and [11S-²H]18:2n ? 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n ? 6 by COX-1 and 18:1n ? 9 by 8R-DOX were both accompanied by large D-KIE (> 20).     

Stereochemistry of lepidopteran sex pheromone biosynthesis: a comparison of fatty acid-CoA Δ11-(Z)-desaturases in Bombyx mori and Manduca sexta female moths

The absolute stereochemistry of fatty acid (FA) desaturation in Bombyx mori and Manduca sexta female pheromone glands (PGs), catalysed by FA-CoA Δ11-(Z)-desaturases, was determined using chiral, specifically labelled palmitic acids {[2,2,3,4,5,5,6,6,7,8,9,9,11,12−²H₁₄]–(11R,12S)−1 and [2,2,3,4,5,5,6,6,7,8,9,9,11,12−²H₁₄]–(11S,12R)−1)} as metabolic probes. The (11R,12S)−1 acid was converted in PGs of treated virgin females to labelled methyl (11Z)-hexadecenoate ([²H₁₄]−2, Mw=282 Da). In incubations with the opposite enantiomer two deuterium atoms from (11S,12R)−1 were removed, yielding [²H₁₂]−2 of Mw=280 Da. These results were confirmed by methylthiolation of [²H₁₄]−2 and [²H₁₂]−2 with a dimethyl disulfide/iodine mixture. Mass spectra of the DMDS adducts directly showed the distribution of deuterium atoms in the labelled methyl esters of 2. The data consistently indicate, that the studied insects possess Δ11-(Z)-desaturases with pro-(R) C(11)-H and pro-(R) C(12)-H stereospecificity, catalysing a syn-elimination of two hydrogen atoms.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Biosynthetic pathways of the sex pheromone components and substrate selectivity of the oxidation enzymes working in pheromone glands of the fall webworm, Hyphantria cunea

The fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae), is a harmful polyphagous defoliator. Female moths produce the following four pheromone components in a ratio of about 5:4:10:2; (9Z,12Z)-9,12-octadecadienal (I), (9Z,12Z,15Z)-9,12,15-octadecatrienal (II), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene (III), and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene (IV). Although ¹³C-labeled linolenic acid was not converted into trienal II at the pheromone glands of H. cunea females, GC-MS analysis of an extract of the pheromone gland treated topically with ¹³C-labeled linolenyl alcohol showed the aldehyde incorporating the isotope. Other C₁₈ and C₁₉ fatty alcohols were also oxidized to the corresponding aldehydes in the pheromone gland, indicating a biosynthetic pathway of IIvia linolenyl alcohol and low substrate selectivity of the alcohol oxidase in the pheromone gland. On the other hand, epoxydiene III was expected to be produced by specific 9,10-epoxidation of the corresponding C₂₁ trienyl hydrocarbon, which might be biosynthesized from dietary linolenic acid in oenocytes and transported to the pheromone gland. The final biosynthetic step in the pheromone gland was confirmed by an experiment using deuterated C₂₁ triene, which was synthesized by the chain elongation of linolenic acid and LiAlD₄ reduction as key reactions. When the labeled triene was administered to the female by topical application at the pheromone gland or injection into the abdomen, deuterated III was detected in a pheromone extract by GC-MS analysis. Furthermore, the substrate selectivity of epoxidase and selective incorporation by the pheromone glands were examined by treatments with mixtures of the deuterated precursor and other hydrocarbons such as C₁₉-C₂₃ trienyl, C₂₁ dienyl, and C₂₁ monoenyl hydrocarbons. The 9,10-epoxy derivative of each alkene was produced, while the epoxidation of the C₂₁ monoene was poorer than those of the trienes and diene. The low selectivity indicated that the species-specific pheromone of the H. cunea female was mainly due to the critical formation of the precursor of each component.