Figure 1.
Phylogenetic analysis of FoxLOX.
FoxLOX was analyzed with different LOXes from plants (Arabidopsis thaliana), mammals (Mus musculus) and mainly putative LOXes from different fungal species (Aspergillus flavus, Aspergillus fumigatus, Botryotinia fuckeliana, Chaetomium globosum, Fusarium oxysporum, Fusarium graminearum, Fusarium verticillioides, Gaeumannomyces graminis, Grosmannia clavigera, Laccaria bicolor, Lentinus sajor-caju, Magnaporthe salvinii, Metarhizium anisopliae, Metarhizium acridum, Myceliophthora thermophila, Nectria haematococca, Neosartorya fischeri, Neurospora crassa, Neurospora tatrasperma, Pleurotus ostreatus, Pleurotus sapidus, Podospora anserina, Rhizoctonia solani, Sordaria macrospora, Thielavia terrestris): A. flavus-LOX1 (AFL2G_06556.2); A. fumigatus-LOX1 (AFUA_4G02770); A fumigatus-LOX2 (Afu7g00860); A. thliana-LOX1 (At1g55020); A. thaliana LOX2 (At3g45140); A. thaliana LOX3 (At1g17420); A. thaliana LOX4 (At1g72520); A. thaliana LOX5 (At3g22400); A. thaliana LOX6 (At1g675609); B. fuckeliana-LOX (CCD50650.1); C. globosum-LOX (XP_001225066.1); F. graminearum-LOX1 (FGSG_02216); F. verticillioides-LOX1 (FVEG_05726.3); F. oxysporum-LOX1 (FOXG_02545.2); F. oxysporum-LOX2 [FoxLOX] (FOXG_04807); F. verticillioides-LOX3 (FVEG_09897.3); F. verticillioides-LOX2 (FVEG_03347.3); G. graminis 13R-MnLOX (AAK81882.1); G. clavigera-LOX (EFX02092.1); L. bicolor-LOX (XP_001881489.1); L. sajor-caju-LOX (CCV01579.1); M. acridum-LOX (EFY84539.1); M. anisopliae-LOX (EFZ04186.1); M. graminicola-LOX (EGP90986.1); M. musculus 5-LOX (NP_033792.1); M. musculus 8-LOX (NP_033791.1); M. musculus 15-LOX (NP_033790.3); M. musculus 12R-LOX3 (NP_035916.1); M. musculus 12S-LOX (P39655.4); M. musculus 12R-eLOX (O70582.1); M. salvinii 9S-MnLOX (CAD61974); M. thermophila-LOX1 (AEO59314.1); M. thermophila-LOX2 (AEO56683.1); N. crassa-LOX (CAD37061.1); N. fischeri-LOX1 (NFIA_030810); N. fischeri-LOX2 (NFIA_113540); N. fischeri-LOX3 (XP_001262720.1); N. haematococca-LOX1 (XP_003042428.1); N. tetrasperma-LOX (EGO61406.1); P. anserina-LOX1 (XP_003437217.1); P.anserina-LOX2 (XP_001911188.1); P. ostreatus-LOX1 (BAI99788.1); P. ostreatus-LOX2 (CCV01578.1); P. sapidus-LOX (CAQ87588.1); R. solani-LOX (CCO32261.1); S. macrospora-LOX (XP_003348064.1); T. terrestris-LOX (AEO63144.1). [35] The phylogenetic tree was constructed by using the ClustalX- software using default parameters. The tree was visualized using tree-view.
Figure 2.
Partial amino acid alignment of different LOX forms with FoxLOX.
Sequences were chosen from F. oxysporum (Fox), A. fumigatus (Af), G. graminis (Gg), A. thaliana (At) and M. musculus (Mm). Alignments were performed using the ClustalX software package employing default parameters. Different determinants for regio- and stereo specificity are indicated as “Bo” (Borngräber according to [66]), “Ho” (Hornung according to [44]), “Sl” (Sloane according to [43]) and “Co” (Coffa according to [45]). Amino acid residues involved in iron binding are indicated by “Li”.
Figure 3.
SDS-PAGE analysis of different fractions of FoxLOX purification.
Shown is a 10% SDS-PAGE analysis of crude cell extract (load, L) applied on an immobilized Ni-column, unbound (flow through, FT), washed proteins (W) and eluted FoxLOX.
Table 1.
Fatty acid composition of F. oxysporum.
Figure 4.
Product specificity of FoxLOX.
SP-HPLC/DAD analysis at 234 nm of products formed from incubation of affinity purified FoxLOX with 18∶2(n-6) (A) 18∶3(n-3) (B) 18∶3(n-6) (C) 20∶4(n-6) (D) for 30–60 min and extractive isolation. Insets show an enlarged version of the respective region of the chromatogram. Before the analysis hydroperoxy fatty acids were reduced to the respective hydroxyl derivatives and pre-purified by RP-HPLC. Products were identified by comparison with authentic standards.
Table 2.
Products formed from incubations of different fatty acid substrates and crude cell extracts of FoxLOX-expressing E. coli Bl21star cells.
Figure 5.
MS analysis of products formed from 18∶3(n-3) and fatty acid hydroperoxidase activity of FoxLOX.
Shown are the MS2 spectra of 12,13-Ep-11-HOD (A), 9,16-DiH(P)OT (B) and 13-KOT derived from incubations of 18∶3(n-3) and FoxLOX.
Table 3.
Kinetic properties of FoxLOX.
Figure 6.
HPLC analysis of the products formed from 18∶2(n-6) (A), 18∶2(n-6)-Me (B) and di18∶2(n-6) PC (C).
Reactions were performed in 50 mM Tris/HCl (pH 7.5) containing 10% glycerol, 0.1% sodium desoxycolate and 300 mM NaCl at room temperature for 60 min. Incubations with 18∶2(n-6) and di18∶2(n-6) PC were methylated or transmethylated, respectively. Before extraction the hydroperoxy fatty acids were reduced to their corresponding hydroxyl derivatives. The analysis was carried out by using SP-HPLC. As insets the CP-HPLC of each product is shown.
Figure 7.
Analysis of the oxygenation mechanism using stereo specifically deuterated 11S-D-18∶2(n-6).
(A) Shown is a progress curve at 234nm of the incubation of 100 µM 18∶2(n-6) and 11S-D-18∶2 (n-6), respectively, and 5 µg affinity purified FoxLOX in 200 µL 50 mM Tris/HCl (pH 8.0). After incubation for additional 30 min the products were extracted and analyzed by using RP-HPLC/MS2. (B) Shown is a structure of 13-HOD (carboxylate anion) with the formation of major fragment ions and (C) an extracted ion chromatogram (XIC) of a mass range of 294.5–295.5 amu with the respective tandem-MS spectra.
Figure 8.
Conversion of 18∶3(n-3) by FoxLOX leading to a set of different products.
The reaction starts with stereo specific abstraction of a specific hydrogen either from the C-11 or C-14 of the fatty acid backbone yielding a carbon centered radical that is delocalized via the π-electron system. In case of the predominant 13-LOX activity the proR-hydrogen is abstracted from the C-11 and oxygen is inserted antarafacially at the C-13 yielding 13S-HPOT. The product is further converted either by isomerization yielding 12,13-Ep-11-HOD or dehydration forming 13-KOT. In case of 9-LOX activity 9-HPOT is formed by hydrogen insertion at the C-9. Abstraction of the hydrogen at the C-14 leads analogously to the formation of a delocalized carbon centered radical that can be either trapped at the C-12 (12-LOX activity) or at the C-16 (16-LOX activity) yielding 12-H(P)OTE or 16-H(P)OTE, respectively. The latter product might be further converted to 15,16-EpOT by elimination of a hydrogen from the C-11. The epoxy-group of this leukotriene A like metabolite can be attacked by water either at the C-15 (yielding 15,16-DiHOT) or at the C-9 (yielding 9,16-DiHOT).