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

Article abstract of DOI:10.1016/j.bbalip.2018.08.012

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.

Full text of DOI:10.1016/j.bbalip.2018.08.012

BBA-MolecularandCellBiologyofLipids1863(2018)1378–1387
Contents lists available at ScienceDirect
BBA - Molecular and Cell Biology of Lipids
journal homepage: www.elsevier.com/locate/bbalip
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
Ernst H. Oliw
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Keywords:
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 un-
saturated 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 de-
rivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.
Dioxygenase-cytochrome P450 fusion proteins
Lipidomics
Lipid metabolism
Mass spectrometry
Oxygenation mechanism
Peroxidase-cyclooxygenase superfamily
1. Introduction
The 3D structures of fatty acids in the active sites have been de-
termined in crystals of COX, α-DOX, and coral 8R-LOX. Ovine COX-1
Polyunsaturated fatty acids can be oxygenated by both dioxygenases
(DOX) of the peroxidase-cyclooxygenase (COX) superfamily and by li-
poxygenases (LOX) [1–3]. The peroxidase-COX superfamily contains
three eukaryotic divisions: the animal COX, the plant α-DOX [4–6], and
the fungal linoleate diol synthase (LDS) families [2]. DOX shares a
common oxygenation mechanism [1,7–9]. With these enzymes, a con-
served Tyr residue is oxidized to a radical, which abstracts hydrogen
from an allylic or bis-allylic carbon or from an α‑carbon. The carbon-
centered radical reacts with molecular oxygen to form a peroxyl radical
[1,7,10], which can be further transformed to prostaglandin endoper-
oxides or hydroperoxy fatty acids.
binds 20:4n-6 and 18:2n-6 with the ω end in the hydrophobic active site
(“tail first”) and with the carboxyl group tethered to Arg120 close to the
entrance [11–13]. In contrast, α-DOX of Oryza sativa oxidizes palmitic
acid at the α‑carbon with the carboxyl group deep in the active site
(“headfirst”), stabilized by ionic interactions with His-311 and Arg-559,
and with Tyr379 positioned close to the α‑carbon [6] (Fig. 1A). In
analogy, 8R-LOX of Plexaura homomalla binds the carboxyl group of
20:4n-6 by ionic interactions with Arg182 and Glu430 at the entrance
and positions the ω end bound to hydrophobic residues in the substrate
channel (“tail first”) [14].
About 20 enzymes of the LDS family have been characterized, but
Abbreviations: AOS, allene oxide synthase; COX, cyclooxygenase; DOX, dioxygenase; EAS, epoxyalcohol synthase; LDS, linoleate diol synthase; LOX, lipoxygenase;
TIC, total ion current; RP, reversed phase; TPP, triphenylphosphine; 8-H(P)ODE, 8-hydro(pero)xy-9Z,12Z-octadecadienoic acid; 18:2n-6-Gly, N-[9Z,12Z-octadeca-
dienoyl]-S-glycine; 18:2n-6-Ile, N-[9Z,12Z-octadecadienoyl]-S-isoleucine; 18:3n-3-Gly, N-[9Z,12Z,15Z-octadecatrienoyl]-S-glycine; 18:3n-3-Ile, N-[9Z,12Z,15Z-oc-
tadecatrienoyl]-S-isoleucine; 8-H(P)OTrE, 8-hydro(pero)xy-9Z,12Z,15Z-octadecatrienoic acid; 9-H(P)ODE, 9-hydro(pero)xy-10E,12Z-octadecadienoic acid; 9-H(P)
OTrE, 9-hydro(pero)xy-10E,12Z,15Z-octadecatrienoic acid; 10-H(P)ODE, 10-hydro(pero)xy-8E,12Z-octadecadienoic acid; 10-H(P)OTrE, 10-hydro(pero)xy-
8E,12Z,15Z-octadecatrienoic acid; 13-H(P)ODE, 13-hydro(pero)xy-9Z,11E-octadecatrienoic acid; 13-H(P)OTrE, hydro(pero)xy-9Z,11E,15Z-octadecatrienoic acid; 16-
H(P)OTrE, 16-hydro(pero)xy-9Z,12Z,14E-octadecatrienoic acid
E-mail address: Ernst.oliw@farmbio.uu.se.
https://doi.org/10.1016/j.bbalip.2018.08.012
Received 21 March 2018; Received in revised form 10 August 2018; Accepted 20 August 2018
Availableonline24August2018
1388-1981/©2018ElsevierB.V.Allrightsreserved.

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A-MolecularandCellBiologyofLipids1863(2018)1378–1387
Fig. 1. Amino acids near the carboxyl group of palmitate in
the crystal structure of α-DOX (Oryza sativa; PDB file 4KVL)
and a model of amino acids of At-8R-DOX-5,8-LDS near C-8 of
linoleate. A. Partial crystal structure of the Tyr379Phe mutant
of α-DOX near the carboxyl group of palmitate. B. Swiss
model of At-8R-DOX-5,8-LDS, based on the crystal structure of
ovine COX-1 with linoleate (PDB file 1IGZ). Linoleate was
positioned in the active site in reverse orientation compared
to the orientation in COX-1 (see text). Val318 is important for
substrate binding and Tyr365 for catalysis, but the Tyr318
residue in the vicinity is not required for catalysis.
the first crystal structure is still pending [8]. The LDS family contains
8-, 9-, and 10-DOX usually fused to catalytically competent cyto-
chromes P450 [15–17]. Electron paramagnetic resonance, site-directed
mutagenesis, and modeling of 8R-DOX-LDS suggest that C-8 is posi-
tioned close to the catalytic Tyr residue (cf. Tyr365 in Fig. 1B)
[7,8,18–20]. The aligned sequence identities between COX and 8R-DOX
domains are unfortunately too low (about 25%) to allow modeling of
the entire substrate channel.
LDS enzymes with 8R-DOX domains characteristically oxidize
18:2n-6 and 18:3n-3 efficiently and 20:2n-6 slowly, but neither 18:3n-6
nor 22:5n-6 [21]. The orientation of fatty acids in the 8R-DOX domains
is difficult to predict from these observations. 9R-DOX of Fusarium
oxysporum of the LDS family oxidizes 22:5n-6 almost as efficiently as
18:2n-6 [22], suggesting tail first orientation in the active site. In
analogy, 13S-LOX-1 of Glycine max (GmLOX-1) oxidizes 18:2n-6, long
chain fatty acids (22:5n-6), and 18:2n-6 of phosphatidylcholine and
lysophosphatidylcholine, which supports “tail first” binding to the ac-
tive site [23–25]. These indirect methods are well suited to study en-
zymes with tail first orientation, but they are often less conclusive for
enzymes with headfirst orientation.
It is likely that fatty acids in headfirst orientation tether the carboxyl
group by ionic interactions with charged residues as illustrated by α-
DOX (Fig. 1A). Fatty acids derivatized with amino acids will retain a
carboxyl group; Gly conjugate will extend the carboxyl end with eH-
NeCH2eCOOH. This will allow ionic interactions with the carboxyl
group to charged residues; the amide group cannot be charged but the
carbonyl may form hydrogen bonds and interact with the protein.
In a recent study, the Ile and Trp conjugates of 18:2n-6 and 18:3n-3
were found to be rapidly oxidized at C-9 by two DOX of F. oxysporum.
Fo-9R-DOX and Fo-9S-DOX-allene oxide synthase (AOS) [26]. In ana-
logy with the lipoxidation of lysophosphatidylcholine by GmLOX-1
[25], these results suggested that fatty acids enter the substrate channel
of Fo-9R-DOX and Fo-9S-DOX-AOS with their ω ends first (Fig. 2A). The
oxidation of Gly, Ile, and Trp conjugates by 8R-DOX domains yielded
unexpected results. 8R-DOX-7,8-LDS of Magnaporthe oryzae (Mo-8R-
DOX-7,8-LDS) oxidized 18:2n-6 and the Gly conjugate rapidly to hy-
droperoxides at C-8 and to 7,8-diols [26]. The Ile and Trp conjugates of
18:2n-6 were not oxidized at C-8. Furthermore, three 8R-DOX-LDS
homologues and 8R-DOX-AOS of Coccidioides immitis (Ci-8R-DOX-AOS)
also oxidized the Gly conjugates at C-8, but not the Ile and Trp con-
jugates at this position [26]. This suggested that five homologous of 8R-
DOX domains bind 18:2n-6 in headfirst orientation for oxidation at C-8
(Fig. 2B). Definitive proof will require the crystal structure of an 8R-
DOX domain with 18:2n-6 in the active site.
respectively. This suggested that fatty acids derivatized with amino
acids might conceivably be used to study oxidation of fatty acids in tail
first position of enzymes, which naturally bind fatty acids headfirst. The
results might reveal catalytic similarities of homologous enzymes with
different substrate orientation, although binding the substrates in the
improper tail first orientation will be expected to reduce the catalytic
turnover.
Based on this hypothesis, the first goal of the present study was to
determine the oxidation of conjugated fatty acids at other positions
than C-8 by 8R-DOX domains. 8R-DOX-5,8-LDS of Aspergillus terreus (At-
8R-DOX-5,8-LDS) [27], 8R-DOX-7,8-LDS of Gaeumannomyces graminis
(Gg-8R-DOX-7,8-LDS) [15,28], Ci-8R-DOX-AOS, and 8S-DOX-AOS of
Zymoseptoria tritici (Zt-8S-DOX-AOS) [29] were chosen for these studies
in an extension of the previous investigation [26]. The sequence simi-
larities of their 8R-DOX domains is indicated by the phylogeny in
Fig. 2C.
Fatty acids derivatized with amino acids are a novel tool to study
enzymatic oxidation, but this method has shortcomings, which are also
important to document. The second goal was to illustrate these lim-
itations with 9R-DOX-AOS of A. niger (An-9R-DOX-AOS) [30]. The 9-
DOX domains of An-9R-DOX-AOS [30] and Fo-9S-DOX-AOS [16] can be
aligned with 54% sequence identity (Fig. 1C). An-9R-DOX-AOS could
therefore be expected to bind fatty acids tail first in analogy with Fo-9S-
DOX-AOS, however, it was found that this was not the case. The fatty
acids derivatized with amino acids were not transformed and provided
little other information, but the classical method of oxidation of sub-
strate analogs (16:2n-4 and 16:2n-6) confirmed headfirst binding to the
active site of An-9R-DOX-AOS.
2. Materials and methods
2.1. Materials
18:2n-6 (99%), 18:3n-3 (99%), S-isoleucine, S-glycine, ethyl chlor-
oformate, triphenylphosphine (TPP), GmLOX-1 (Lipoxidase), and trie-
thylamine were from Sigma. 16:2n-6, 16:2n-4 and 20:2n-6 were from
Larodan (Solna, Sweden). [13C18]18:2n-6 (98% 13C; 95%) was from
Cambridge Isotope Laboratories (Andover, MA, USA). 9S- and 9R-hy-
droperoxy-10E,12Z,15Z-octadecatrienoic acid (9-HPOTrE) and 9S-hy-
droperoxy-10E,12Z-octadecadienoic acid (9-HPODE) were obtained by
biosynthesis (9S-LOX of tomato [31] and recombinant 9R-LOX of
Anabaena sp. PCC 7120 [32]). The amino acid conjugates of fatty acids
were prepared as described [26,33]. The same method was used to
prepare conjugates of 9S- and 9R-HOTrE. [2H,11S]18:2n-6 (99% 2H),
[2H,11R]18:2n-6 (25% 2H), and 9S-hydroxy-10R(11R)epoxy-12Z-octa-
decenoic acid from Larodan were derivatized with Gly in the same way.
The ester derivative of the hydroxy group of 9-HOTrE conjugates was
regenerated by alkaline hydrolysis (0.5 M KOH in methanol with 10%
water under Ar; 70 °C, 1 h). Rac HPOTrE-Ile, rac HPODE-Ile, and rac
An intriguing observation was that Ile and Trp conjugates of C18
fatty acids appeared to be mainly oxidized at C-9 by several 8R-DOX
domains [26], possibly by binding in tail first position. In addition, 8R-
DOX domains could also oxidize the corresponding Gly conjugates at C-
8 and C-9; apparently by hydrogen abstraction at C-8 and C-11,
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Fig. 2. Overview of products formed from 18:3n-3 by Fo-9S-
DOX-AOS, Gg-8R-DOX-7,8-LDS, and Ci-8R-DOX-AOS and se-
quence similarities of three pairs of 8- and 9-DOX domains. A.
Overview of the sequential biosynthesis of 9S-hydroperoxy-
10E,12Z,15Z-octadecatrienoic acid (9S-HPOTrE) and an al-
lene oxide by Fo-9S-DOX-AOS. B. Overview of the sequential
biosynthesis of 8R-hydroperoxy-9Z,12Z,15Z-octadecatrienoic
acid (8R-HPOTrE) and a 5,8-diol or an allene oxide by At-8R-
DOX-7,8-LDS and Ci-8R-DOX-AOS, respectively. C.
Phylogenetic tree constructed from the DOX domains of three
homologous pairs: 9S-DOX-AOS (EGU88194) and 9R-DOX-
AOS (EHA25900); 8S-DOX-AOS (EGP83657) and 8R-DOX-
AOS (EAS28473); 8R-DOX-5,8-LDS (ATEG_03992) and 8R-
DOX-7,8-LDS (Q9UUS2). The DOX domains were defined as
the N-terminal sequences, which aligned with 700 N-terminal
amino acids of Gg-8R-DOX-7,8-LDS. The aligned amino acid
identity of the enzyme pairs in is indicated in percent.
HPODE-Gly were obtained by photooxidation. 3 mg of the conjugates in
0.6 ml methanol with 160 μM methylene blue were flushed with O2 and
exposed to light for 3–4 h [34]. The hydroperoxides were then im-
mediately reduced to alcohols by treatment with excess NaBH4 (4 °C).
Cartridges with octadecyl silica (Sep-Pak Classic C18) were from Waters
(Baltimore, MD, USA). Yeast extract and peptone from casein were from
VWR International (Spånga, Sweden).
and New England Biolabs, Ipswich, MA, USA).
The cells were lysed as described [15,16,27,29,35]. The lysates were
centrifuged (17,000 ×g; 20 min; 4 °C) and aliquots of 1.8 ml were
stored at −80 °C until enzyme assay. The expression of An-9R-DOX-
AOS shows that lysates of E. coli do not oxidize fatty acids derivatized
with Gly or Ile (see below). All enzymes preparations were confirmed to
metabolize 18:2n-6 as previously reported [15,16,27,29,35].
The recombinant enzymes in 0.5–2.0 ml 0.1 M KHPO4 (pH 7.4)/
2 mM EDTA/0.04% Tween-20 were usually incubated with 100 μM
fatty acid conjugates for 30–40 min on ice. The reaction was terminated
by addition of water (10 ml) and extractive isolation was performed
with Sep-Pak/C18 cartridges, which were eluted with 4 ml ethyl acetate.
The organic phase was evaporated to dryness (N2) and the hydroper-
oxides were reduced to alcohols by treatment with TPP in hexane. The
samples were dried (N2) and then dissolved in 50–200 μl ethanol or
hexane/isopropanol, 90/10, for analysis by LC-MS.
2.2. Enzyme expression and assay
At-8R-DOX-5,8-LDS (AGA95448; ATEG_03992), a splice variant of
At-8R-DOX-5,8-LDS (ATEG_03992_sv; without functional cytochrome
P450 domain), Gg-8R-DOX-7,8-LDS (Q9UUS2), Ci-8R-DOX-AOS
(EAS28473), Zt-8S-DOX-AOS (EGP83657), Fo-10R-DOX-epoxyalcohol
synthase (EAS) (EGU86021) and An-9R-DOX-AOS (EHA25900) were
expressed in Escherichia coli BL21 cells (Invitrogen, Waltham, MA, USA
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2.3. LC-MS analysis and characteristic fragments
At-8R-DOX-5,8-LDS oxidized 18:2n-6-Gly to H(P)ODE and to epox-
yalcohols. Steric analysis showed that the S stereoisomers of 9- and 13-
HODE-Gly predominated (75%). The epoxy alcohols were the major
products and were separated into a major and a minor peak (marked I
and II in Fig. 3B). These epoxyalcohols could be formed from the hy-
droperoxides non-enzymatically or by the 5,8-LDS domain, but we
could not detect transformation to the Gly conjugate of 5,8-DiHODE.
The MS/MS spectrum (m/z 368 → full scan) of the main epox-
yalcohol in peak I showed strong signals at m/z 228 (A−-140) and 258
(228 + 30). This fragmentation was consistent with the Gly conjugate
of 9(10)epoxy-11-hydroxy-12Z-octadecenoic acid (cf. [37]). The epox-
yalcohol, which eluted on the right shoulder of peak I showed signals,
inter alia, at m/z 254 (A−-114) and 268 (A−-100), compatible with the
Gly derivative of 12(13)epoxy-11-hydroxy-9Z-octadecenoic acid. The
second eluting peak (II) showed signals at m/z 214 (A−-124; 100%),
228 (50%), and 244 (214 + 30; 30%), suggesting a Gly conjugate of
8(9)epoxy-10-hydroxy-12Z-octadecenoic acid [37].
At-8R-DOX-5,8-LDS oxidized 18:3n-3-Gly essentially as 18:2n-6 to
8(9)- and 9(10)epoxyalcohols, but the Gly derivative of 16-hydro(pero)
xy-9Z,12Z,14E-octadecatrienoic acid (16-HPOTrE-Gly) was the main
hydroperoxide. The latter was identified (after reduction) by a MS/MS
spectrum (m/z 350 → full scan) with the characteristic loss of
HCOeCH2eCH3, which yielded a strong signal at m/z 292 (A−-58). The
relative amounts of 8-, 9-, 13- and 16-HOTrE-Gly were 0.7:0.2:0.3:1,
respectively, but these numbers are not corrected for the transformation
to epoxyalcohols. 13-HOTrE-Gly was estimated by the characteristic
and intense ion at m/z 253 (350-97), whereas 13-HODE-Gly yields the
corresponding ion at m/z 252 (352-100) [26].
RP-HPLC with MS/MS analysis was performed with a Surveyor MS
pump (ThermoFisher, Waltham, MA, USA) and an octadecyl silica
column (5 μm; 2 × 150 mm; Phenomenex, Torrance, CA, USA), which
was eluted at 0.3 ml/min with methanol/water/acetic acid, 700/300/
0.05, 750/250/0.05, or 800/200/0.05 (v/v/v).
The effluent was subject to electrospray ionization in a linear ion
trap mass spectrometer (LTQ, ThermoFisher) equipped with the Ion
Max Source (ThermoFisher). The heated transfer capillary was set at
315 °C, the ion isolation width at 1.5 amu or 3 amu, the collision energy
at 35 (arbitrary scale), and the tube lens at about −110 V.
Prostaglandin F1α (The Upjohn Co., Kalamazoo, MI, USA) was infused
for automatic tuning of the signal at m/z 355. Samples were injected
manually (Rheodyne 7510; Rheodyne, Bensheim, Germany) or by an
auto sampler (Surveyor Autosampler Plus, ThermoFisher).
CP-HPLC-MS/MS was performed with Reprosil Chiral AM (5 μ;
2 × 300 mm; Dr. Maisch, Ammerbuch, Germany), which was eluted
(0.2 ml/min)
with
hexane/methanol/ethanol/acetic
acid,
80:10:10:0.02 or 90:5:5:0.02 (v/v/v/v) for analysis of HODE-Ile/
HOTrE-Ile and HODE/HOTrE-Gly, respectively. The eluate was mixed
on-line with 2-propanol/water, 3/2 (v/v), at 0.15 ml/min from a
second pump (Constametric 3200, LDC/MiltonRoy, Houston, TX, USA).
The combined effluents were introduced by electrospray ionization into
the ion trap mass spectrometer above.
MS/MS spectra were recorded by fragmentation of the carboxylate
anions and analyzed by reconstructed ion chromatograms. The Gly and
Ile derivatives of hydroxy fatty acids were identified by characteristic
fragment ions formed by specific losses from the carboxylate anions
(A−) as described [26]. Amino acid derivatives of 8-hydroxy-9Z,12Z-
3.1.2. Oxidation of [2H,11S]- and [2H,11R]18:2n-6-Gly
octadecadienoic acid (8-HODE) show a characteristic fragment at A−
-
8R-DOX domains oxidize 18:2n-6 by antarafacial hydrogen ab-
straction and oxygen insertion at C-8 [38]. It is likely that 8R-HPODE-
Gly is also formed in this way. We next examined whether At-8R-DOX-
5,8-LDS oxidized 18:2n-6-Gly to 9-HPODE-Gly by antarafacial hy-
drogen abstraction at C-11 and oxygenation at C-9. Steric analysis of 9-
HODE-Gly by CP-HPLC showed that it contained 75% of the S stereo-
isomer (see below).
138, 9-HODE at A−-124, 10-hydroxy-8E,12Z-octadecadienoic acid (10-
HODE) at A−-112, and 13-hydroxy-9Z,11E-octadecadienoic acid (13-
HODE) at A−-100 [36]. The Gly and Ile derivatives of 13-hydroxy-
9Z,11E,15Z-octadecatrienoic acid (13-HOTrE) showed signals at A−-97
[26]. In the same way we used characteristic ions of the MS/MS spectra
of epoxyalcohols to identify their Gly and Ile derivatives. The metabolic
profile of the different enzymes reported here was based on product
analysis and fragmentation during LC-MS/MS analysis. The ion in-
tensities of these metabolites only provide relative information and
absolute levels must be confirmed in the future with more reliable
methods.
[2H,11S]18:2n-6-Gly (99% 2H) was oxidized by At-8R-DOX-5,8-LDS
to epoxyalcohols, which were separated into two peaks by RP-HPLC as
discussed above (cf. peaks I and II in Fig. 2B). The 9(10)epoxyalcohol in
the first peak retained most of the 2H label (Fig. 2C) as judged from the
isotope distribution in the A−-18 fragment with protium or with deu-
terium; ratio (m/z 350)/(m/z 351) = 0.29, which suggested that 78% of
the 2H label was retained. The [2H,11S] label was also retained in 9S-
HODE-Gly to the same extent (CP-HPLC analysis). The signal at m/z 350
due to loss of 2H label was 6 times larger in the mass spectrum of the
9(10)epoxyalcohol than the 8(9)epoxyalcohol of this experiment
(Fig. 2C). The deuterium/protium ratio (m/z 350)/(m/z 351) of the
8(9)epoxyalcohol was 0.05 (Fig. 2C), which confirmed the expected
retention of the 2H label at C-11. Furthermore, [2H,11R]18:2n-6-Gly
(25% 2H) was oxidized with significant loss of the 2H label in the Gly
derivative of the 9(10)epoxyalcohol (70%) when compared to the 2H
content of the corresponding Gly derivative of the 8(9)epoxyalcohol.
We conclude that 9S-HPODE-Gly (and the 9(10)epoxyalcohol) were
biosynthesized by At-8R-DOX-5,8-LDS and to a large extent (about
75%; cf. Fig. 3C) by abstraction of the proR hydrogen at C-11 and an-
tarafacial oxygenation at C-9.
2.4. Miscellaneous
The concentration of derivatized fatty acids was determined by
oxidation of an aliquot with GmLOX-1 in 0.1 M NaBO3 (pH 9.0) and UV
spectra were recorded with a dual beam spectrophotometer (Shimadzu
UV-PC-2101). Phylogenetic trees were constructed with the ClustalW
algorithm (Lasergene software, DNASTAR Inc., Madison, WI, USA).
SWISS-MODEL (Biozentrum, Basel, Switzerland) was used for modeling
and PyMOL Molecular Graphics System (Version 1.7.4 Schrödinger,
LLC) for further analysis.
3. Results
3.1. At-8R-DOX-5,8-LDS
3.1.1. Oxidation of 18:2n-6-Gly and 18:3n-3-Gly
3.1.3. Oxidation 18:2n-6-Ile and 18:3n-3-Ile
The expression construct of a splice variant of At-8R-DOX-5,8-LDS
without functional cytochrome P450 domain (ATEG _03992_sv [27])
oxidized 18:2n-6-Gly to 8-, 9-, and 13-H(P)ODE-Gly in a ratio of
0.3:1:0.7, respectively (Fig. 3A). 8-, 9- and -Gly were analyzed as al-
cohols by the characteristic signals at m/z 214, 228, and 252, respec-
tively, as discussed above. Only traces of epoxyalcohols were formed
from the hydroperoxides.
At-8R-DOX-5,8-LDS oxidized 18:2n-6-Ile to 9-H(P)ODE-Ile and 13-H
(P)ODE-Ile in a ratio of 1:0.5 (Fig. 4A). Steric analysis showed that 77%
of the 9S stereoisomer was formed, but 13-HODE-Ile was almost ra-
cemic (Fig. 4B). It is noteworthy that 13-HODE-Ile and 13-HOTrE-Ile
form characteristic ions at m/z 308 (408-100) and m/z 309 (406-97),
respectively [26].
At-8R-DOX-5,8-LDS oxidized 18:3n-3-Ile to 9- and 16-HOTrE-Ile in a
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Fig. 3. LC-MS/MS analysis of hydroperoxides and epoxyalcohols formed from 18:2n-6-Gly by At-8R-DOX-5,8-LDS. The hydroperoxides were reduced to alcohols with
TPP before analysis. A. RP-HPLC-MS/MS analysis of HODE-Gly (m/z 352 → full scan). Top, selected ion monitoring of 8-HODE-Gly (m/z 214); middle, selected ion
monitoring of 9-HODE-Gly (m/z 228); bottom, selected ion monitoring of 13-HODE-Gly (m/z 252). B. RP-HPLC-MS/MS analysis of diols and epoxyalcohols (m/z
368 → full scan) formed from 18:2n-6-Gly. Top trace, TIC; middle trace, selected ion monitoring of 8(9)epoxyalcohols-Gly (m/z 214); lower trace, selected ion
monitoring of 9(10)epoxyalcohols-Gly (m/z 228). C. Partial mass spectra of 9(10)- and 8(9)epoxyalcohols formed from [2H,11S]18:2n-6-Gly. Left, partial MS/MS
spectrum of the [2H]9(10)epoxyalcohol centered at m/z 351 (A−-18). Right, partial MS/MS spectrum of [2H]8(9)epoxyalcohol-Gly centered at m/z 351 (A−-18). The
2H label was partly lost in the 9(10)epoxyalcohols as shown by the signal intensity at m/z 350.
characteristic signal at m/z 292 (A−-58; loss of HCOeCH2eCH3). 16-
HOTrE-Ile eluted in a single peak from the CP-HPLC column (Reprosil
Chiral AM), which separates fatty acid stereoisomers of cis-trans con-
jugated fatty acids. The structure of this stereoisomer was not de-
termined.
3.2. Gg-8R-DOX-7,8-LDS
3.2.1. Oxidation of 18:2n-6-Gly and 18:3n-3-Gly
Gg-8R-DOX-7,8-LDS oxidize 18:2n-6-Gly at C-8 as previously re-
ported [26], but also at C-9 and C-13 (ratio 1:0.1:0.2, respectively).
Steric analysis showed that 83% of the 9S-HODE-Gly stereoisomer was
formed, whereas 13-hydroperoxy-9Z,11E-octadecadienoyl-Gly (13-
HPODE-Gly) was almost racemic (42% 13R-HODE-Gly)(Fig. 6A). For
comparison, At-8R-DOX-5,8-LDS formed 75% of the 9S-HODE-Gly ste-
reoisomer (insert in Fig. 6A).
18:3n-3-Gly was oxidized to an 8(9)epoxyalcohol as a major product
and to small amounts 9-, 16-, and 13-H(P)ODE-Gly (Fig. 6B). The
epoxyalcohol was identified as 8(9)epoxy-10-hydroxy-12Z,15Z-octade-
cadienoic acid by the MS/MS spectrum in Fig. 6C. Important signals
were noted at m/z 214 (A−-124), 244 (214 + 30; cf. insert in Fig. 6C),
228, 200 (244-40; possibly loss of CO2) and 170 (214-44). This complex
fragmentation is likely due to Payne rearrangement of 8(9)epoxy-10-
hydroxyoctadecadienoic acid in the mass spectrometer [37].
3.2.2. Oxidation of 18:2n-6-Ile and 18:3n-3-Ile
Fig. 4. LC-MS/MS analysis of products formed from 18:2n-6-Ile by At-8R-DOX-
Gg-8R-DOX-7,8-LDS oxidized 18:2n-6-Ile to 9- and 13-HODE-Ile and
5,8-LDS. The hydroperoxides were reduced to alcohols with TPP before ana-
to 9(10)- and 12(13)epoxyalcohols. CP-HPLC analysis showed that 62%
lysis. A. RP-HPLC-MS/MS analysis of hydroxy metabolites of 18:2n-6-Ile (m/z
of the 9S-HODE-Ile stereoisomer was formed, but 13-HODE-Ile was
408 → full scan). Upper trace, TIC; middle trace, selected ion monitoring of m/z
racemic. 18:3n-3-Ile was transformed to 9-, 13-, and 16-HOTrE-Ile in a
284 (A−-124; 9-HODE-Ile); bottom trace, selected ion monitoring of m/z 308
ratio of 0.4:0.3:1, respectively, as judged from the ion intensities of m/z
284, 309, and 348.
(A−-100; 13-HODE-Ile). B. CP-HPLC-MS/MS analysis of stereoisomers of 9- and
13-HODE-Ile with selected ion monitoring of m/z 284 and 308, respectively.
Integration of the area of the two pairs of peaks suggested that the R and S
stereoisomers were formed in a ratio of 23:77 and 54:46, respectively.
3.3. Ci-8R-DOX-AOS
ratio of 1:0.8, and to smaller amounts of 13-HOTrE-Ile (Fig. 5A). CP-
HPLC analysis showed they consisted of 57% of the 9S-HPOTrE-Ile and
49% of the 13S-HOTrE-Ile stereoisomers. The elution order (9R before
9S) was determined with authentic standards.
16-HOTrE-Ile was identified by the MS/MS spectrum (m/z 350 →
full scan), which is shown in Fig. 5B. The mass spectrum showed the
3.3.1. Oxidation of 18:2n-6-Gly and 18:3n-3-Gly
Ci-8R-DOX-AOS oxidized 18:3n-3-Gly at mainly C-8 as previously
reported [26], but also at C-16, C-13, and C-9 (ratio 1.0:0.1:0.3:0.3,
respectively). 18:2n-6-Gly was also mainly oxidized at C-8; only small
amounts of 9-and 13-HODE-Gly were detected. Biosynthesis of α-ketols
or epoxyalcohols could not be detected.
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Fig. 5. LC-MS/MS analysis of oxidation of 18:3n-
3-Ile by At-8R-DOX-5,8-LDS. Hydroperoxides
were reduced to alcohols with TPP before ana-
lysis. A. RP-HPLC-MS/MS analysis of 9- and 16-
HOTrE-Ile. Upper trace, TIC (m/z 406 → full
scan); second trace, selected ion monitoring of
m/z 284 (A−-122; 9-HOTrE-Ile); third trace, m/z
348 (16-HOTrE-Ile); bottom trace, selected ion
monitoring of m/z 309 (13-HOTrE-Ile). B. MS/
MS spectrum (m/z 406 → full scan) of 16-
HOTrE-Ile. The inset shows formation of m/z 130
(isoleucine) and 348 (406-58, loss of
OCHeCH2eCH3).
3.3.2. Oxidation of 18:2n-6-Ile and 18:3n-3-Ile
3.4. Zt-8S-DOX-AOS
Ci-8R-DOX-AOS oxidized 18:2n-6-Ile to the Ile conjugates of 9-
HODE and 10-hydroperoxy-8E,12Z-octadecadienoic acid (10-HPODE),
as shown in Fig. 7A, and to 13-HODE-Ile. Steric analysis of 9-HODE-Ile
showed that it was almost racemic. 10-HPODE-Ile was apparently
formed by hydrogen abstraction at C-8. The MS/MS spectrum (m/z
408 → full scan) of 10-HODE-Ile is shown in Fig. 7B with informative
signals at m/z 296 (A−-112) and 252 (296-44). C-8 was not oxidized.
18:3n-3-Ile was oxidized to 9-, 13-, and 16-HOTrE in a ratio of
0.6:0.3:1. Only small amounts of 10-HOTrE-Ile were detected (less than
10% of 9- and 13-HOTre-Gly)
3.4.1. Oxidation of 18:3n-3-Gly and 18:3n-3-Ile
18:3n-3-Gly was oxidized at C-16 and C-9, but the oxidation at C-8
was relatively minor. It therefore seems plausible that 18:3n-3-Gly was
oxidized only in tail first position. Zt-8S-DOX-AOS oxidized 18:3n-3-Ile
mainly at C-16 and C-9 in analogy with Ci-8R-DOX-AOS; this similarity
did not merit further confirmation.
Fig. 6. LC-MS/MS analysis of metabolites formed from
18:2n-6-Gly and 18:3n-3-Ile by Gg-8R-DOX-7,8-LDS. The
hydroperoxides were reduced to alcohols with TPP before
analysis. A. CP-HPLC separation of stereoisomers of 9-
and 13-HODE-Gly and on-line MS/MS analysis (m/z
352 → full scan) with selected ion monitoring. Top trace,
m/z 228 (9-HODE-Ile); bottom trace, m/z 252 (13-HODE-
Ile). The inset shows steric analysis of 9-HODE-Gly
formed by 8R-DOX-5,8-LDS. B. RP-HPLC-MS/MS analysis
of products formed from 18:3n-3-Gly by 8R-DOX-7,8-LDS.
Top, total ion current (m/z 366 → full scan; m/z 350 →
full scan); middle, m/z 214 (8-HOTrE-Gly); bottom, m/z
228 + 292 (9- and 16-HOTrE-Gly). C. Mass spectrum of
8(9)epoxy-10-hydroxy-12Z,15Z-octadecadienoyl-Gly.
Due to Payne rearrangement, 8(9)epoxy-10-hydroxy is in
equilibrium with 9(10)epoxy-8-hydroxy in the mass
spectrometer [37], which explains the signals at m/z 244
(cf. insert) and 214 (A−-124).
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Fig. 7. LC-MS/MS analysis of hydroperoxides
formed from 18:2n-6-Ile by Ci-8R-DOX-AOS. The
hydroperoxides were reduced to alcohols with TPP
before analysis. A. RP-HPLC-MS/MS analysis of
HODE-Ile. Upper trace, TIC (m/z 408 → full scan);
middle trace, selected ion monitoring of m/z 284
(A−-(112 + 44); 9-HODE-Ile); bottom trace, se-
lected ion monitoring of m/z 252 (A−-156; 10-
HODE-Ile). B. MS/MS spectrum (m/z 406 → full
scan) of 10-HODE-Ile. The inset shows formation of
two characteristic ions, m/z 296 (A−-112) and m/z
252 (A−-(112 + 44)).
3.5. An-9R-DOX-AOS
9(10)epoxy-11-hydroxy- and 9(10)epoxy-13-hydroxyoctadecenoic
acids [37]. The MS3 spectrum (m/z 283 → 265 → full scan) of the α-
ketol, 9-hydroxy-10-oxo-12Z-hexadecenoic acid, is shown in Fig. 8B.
Important signals were noted at m/z 247 (265-18), 237 (265-28; loss of
CO), 221 (265-44; loss CO2), and in the lower mass range m/z 153, 127,
and 125. The MS/MS spectrum of 9-hydroxy-10E,12Z-hexadecadienoic
acid was analogous to the MS/MS spectrum of 9-HODE.
These results suggested that 18:2n-6 and 16:2n-4 were oxidized in
the same way by the 9R-DOX domain, likely with headfirst orientation.
The relative rate of oxidation of 100 μM 16:2n-4 compared to 100 μM
18:2n-6 was 24%.
3.5.1. Oxidation of 18:2n-6-Gly, 18:2n-6-Ile, and 16:2n-6-Gly
An-9R-DOX-AOS did not oxidize 18:2n-6-Gly and 18:2n-6-Ile sig-
nificantly. Only traces of rac HODE-Ile were found. We next examined
16:2n-6-Gly as a short carbon chain analogue to 18:2n-6-Gly, but it was
not oxidized. These experiments confirmed that cell lysates of E. coli do
not oxidize these conjugated fatty acids in a significant way.
3.5.2. Oxidation of 16:2n-6 and 16:2n-4
Previous work showed that An-9R-DOX-AOS oxidizes 18:2n-6 effi-
ciently but 20:2n-6 and 18:3n-3 at relative rates of only 16 and 5%,
respectively [30]. 18:3n-6 and 18:1n-9 were not oxidized [30]. To de-
termine whether the carbon chain length from C-1 to C-11 or from C-11
to C-18 of 18:2n-6 were critical for catalysis, we investigated 16:2n-6
and 16:2n-4.
An-9R-DOX-AOS oxidized 7Z,10Z-16:2 (16:2n-6) to only traces of 7-
and 11-hydroxy metabolites. In contrast, 9Z,12Z-16:2 (16:2n-4) was
oxidized in analogy with 18:2n-6 to 9-hydro(pero)xy-10E,12Z-hex-
adecadienoic acid and an α-ketol. 9(10)Epoxyalcohols were also
formed and they eluted before the α-ketol on RP-HPLC (Fig. 8A). The
MS/MS spectrum (m/z 282 → full scan) of the first eluting material
showed signals at m/z 171, 201, and 211, which suggested a mixture of
3.6. Fo-10R-DOX-EAS
3.6.1. Oxidation of 16:2n-4 and 16:2n-6
Fo-10R-DOX-EAS abstracts hydrogen at C-8 and inserts oxygen at C-
8 of 18:1n-9 and C-8 and C-10 of 18:2n-6 and 18:3n-3 [35,39]; its DOX
activities are thus closely related to 8R-DOX. This enzyme is included to
illustrate the oxidation of 16:2n-4 and 16:2n-6 in opposite head-to-tail
orientations.
RP-HPLC-MS/MS analysis showed that Fo-10R-DOX-EAS oxidized
16:2n-4 extensively at C-8 and C-10 in analogy with 18:2n-6. In con-
trast, 16:2n-6 was oxidized at a relative rate of only 2% in comparison
with 16:2n-4. Oxidation of 16:2n-6 occurred mainly at C-7, presumably
by oxidation in tail first position.
4. Discussion
This study is an extension of the foregoing investigation on fatty
acids derivatized with amino acids [26]. Five 8R-DOX homologues
oxidized 18:2n-6-Gly either exclusively or significantly at C-8, but not
the Ile and Trp derivatives. The present investigation focuses on oxi-
dation of other carbons than C-8. One of the main findings is that At-8R-
DOX-5,8-LDS oxidizes C-9 of 18:2n-6-Gly in tail first position to 9S-
hydroperoxy metabolites (Fig. 9A). This occurs mainly by antarafacial
hydrogen abstraction at C-11 and O2 insertion at C-9 of 18:2n-6-Gly.
Oxidation of 18:2n-6-Gly and 18:3n-3-Gly illustrates at least two
groups of 8R-DOX domains (Table 1). The first group oxidizes these Gly
derivatives by hydrogen abstraction at mainly or exclusively at C-8
(headfirst) and to a minor extent at C-11 (tail first with O2 insertion at
C-9 and C-13) as illustrated by Gg-8R-DOX-7,8-LDS, Ci-8R-DOX-AOS
and Mo-8R-DOX-7,8-LDS. The latter oxidizes 18:2n-6-Gly and 18:3n-3-
Gly only by hydrogen abstraction and O2 insertion at C-8 [26]. In
contrast, At-8R-DOX-5,8-LDS of the second group abstracts hydrogen at
C-11 of 18:2n-6-Gly,18:3n-3-Gly, 18:2n-6-Ile, and 18:3n-3-Ile with for-
mation of 9-hydroperoxy metabolites as major products (Table 1). Gg-
Fig. 8. LC-MS/MS analysis of the oxidation of 16:2n-4 by An-9R-DOX-AOS. A.
RP-HPLC separation of metabolites and MS/MS and MS3 analysis (m/z 267 and
283). Top trace, total ion current. The first eluting peak contained 9(10)epox-
yalcohols, the second an α-ketol, and the third 9-hydroxy-10E,12Z-hex-
adecadienoic acid. Bottom trace, MS/MS analysis (m/z 267 → full scan) of hy-
droxyhexadecadienoic acids. 9-Hydroxy-10E,12Z-hexadecadienoic was the
main component in the third peak (marked 9-HO-16:2). B. MS/MS spectrum
(m/z 283 → 265 → full scan) of the α-ketol, 9-hydroxy-10-oxy-12Z-hex-
adecenoic acid.
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Table 1
Overview of the oxidation of Gly and Ile conjugates of 18:2n-6 and 18:3n-3 by
8R-DOX domains.
Enzyme
Position of oxidation of 18:2n-6-Gy
C-8
C-9
C-13
At-8R-DOX-5,8-LDS
Gg-8R-DOX-7,8-LDS
Ci-8R-DOX-AOS
+
+++
+
(+)
−
+
+
+
−
+++
+++
+++
Mo-8R-DOX-7,8-LDSa
Enzyme
Position of oxidation of 18:3n-3-Gly
C-8
C-9
C-13
C-16
At-8R-DOX-5,8-LDS
Gg-8R-DOX-7,8-LDS
Ci-8R-DOX-AOSa
+++
+++
+++
+++
+++
+
+
+
(+)
+
+
++
+
Mo-8R-DOX-7,8-LDSa
−
−
(+)
Enzyme
Position of oxidation of 18:2n-6-Ile
C-8
C-9
C-10
C-13
At-8R-DOX-5,8-LDS
Gg-8R-DOX-7,8-LDS
Ci-8R-DOX-AOS
−
−
−
+++
+++
+
−
−
+++
++
+++
+
Enzyme
Position of oxidation of 18:3n-3-Ile
C-8
C-9
C-13
C-16
At-8R-DOX-5,8-LDS
Gg-8R-DOX-7,8-LDS
Ci-8R-DOX-AOS
−
−
−
+++
+
++
+
+
+
+++
+++
+++
The data show semi quantitative LC-MS/MS analysis of Gly and Ile derivatives
of HODE and HOTrE, based on reconstructed ion chromatograms of char-
acteristic fragment ions and their signal intensities. The relative amounts of
metabolites were marked as follows: +++ for the most abundant metabolites
(100–70%); ++ for 69–40%; + for 39–10%; (+) for less than 10%; − for
undetectable or insignificant amounts.
a
Fig. 9. Overview of the stereospecific oxidation of 18:2n-6-Gly in tail first po-
sition by At-8R-DOX-5,8-LDS and a hypothetical pathway of evolution from an
ancestor of 8R-DOX to 9S-DOX. A. Biosynthesis of 9S- and 9R-HPODE-Gly from
18:2n-6-Gly by mainly antarafacial hydrogen abstraction at C-11 and oxygen
insertion at C-9 in seeming analogy with Fo-9S-DOX-AOS. B. According to the
hypothesis, an ancestor of 8R-DOX may have been modified during evolution to
bind 18:2n-6 in the opposite orientation to become a precursor of 9S-DOX. The
latter is then converted to present day 9S-DOX by evolution.
Data from Ref. 24.
This percentage is too low for modeling of the substrate channel based
on the crystal structures of ovine COX-1 with 18:2n-6 or α-DOX of O.
sativa with 16:0 [6,13]. Lastly, 8R-DOX oxidizes 18:2n-6 and 18:3n-3,
but not 18:3n-6. This suggests that the carbon chain from C-1 to C-8
must be saturated to allow hydrogen abstraction at the allylic carbon
(C-8). At-8R-DOX-5,8-LDS could therefore be anticipated to oxidize
18:2n-6-Ile in tail first position by hydrogen abstraction at the allylic
position (C-14) and not at the bis-allylic position (C-11). This proved to
be incorrect.
Fo-9S- and An-9R-DOX-AOS can be aligned with 52% sequence
identity over the 9-DOX domains (Fig. 1). We therefore investigated
whether An-9R-DOX-AOS also oxidized fatty acids in tail first position.
18:2n-6-Gly, 18:2n-6-Ile, and 16:2n-6-Gly were not oxidized and pro-
vided no other information. Shortening of the chain length of 18:2n-6
with two carbons to 7Z,10Z-16:2 (16:2n-6) reduced catalysis almost
completely, but shortening from the ω-end was possible. 9Z,12Z-16:2
(16:2n-4) was oxidized at C-9 and further converted to an α-ketol and
epoxyalcohols. The length of the carbon chain from the carboxyl group
to the bis-allylic hydrogen at C-11 appeared to be critical. The carbon
chain from C-1 to C-8 must also be saturated as neither 18:3n-6 [30] nor
16:2n-6 were oxidized. These observations could be consistent with
headfirst orientation in the active site in analogy with 8R-DOX, which
raises the question of the fungal evolution of 8R-DOX-LDS and 9R-DOX-
AOS on one hand as well as 9S-DOX-AOS.
8R-DOX-7,8-LDS also preferentially metabolizes C-9 of 18:2n-6-Ile, but
Ci-8R-DOX-AOS unexpectedly catalyzed hydrogen abstraction at C-8
with formation of 10-HPODE-Gly (but not 8-HPODE-Gly). This enzyme
likely positioned 18:2n-6-Ile (tail first) deeper in the active site than the
other enzymes. All enzymes oxidize the 18:3n-3 derivatives at C-16, but
linolenoate 16-DOX of the LDS family has not been described.
Hydrogen abstraction at C-11 and oxidation of C-9 suggests simi-
larities with the 9-DOX activities of Fo-9S-DOX-AOS; hydrogen ab-
straction at C-11 and oxidation of C-13, catalyzed by 13-DOX of the LDS
family, has not yet been described. Fo-9S-DOX-AOS is stereo and posi-
tion specific, but it uses the same antarafacial hydrogen abstraction and
oxygenation mechanism as 8R-DOX [30]. Can the oxidation of 18:2n-6-
Gly and 18:2n-6-Ile to 9S-hydroperoxy metabolites be predicted as a
direct consequence of the sequence similarities of At-8R-DOX-5,8-LDS
and Fo-9S-DOX-AOS?
This seems unlikely. Firstly, the amino acids directing headfirst or
tail first positioning of fatty acids are unknown. Secondly, Fo-9S-DOX-
AOS and the three 8R-DOX homologues share 40% sequence identity
(Fig. 1C) and align with 25% sequence identity to COX-1 and α-DOX.
The subfamily of 8R-DOX-LDS is widely distributed in both
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ascomycetous and basidiomycetous fungi according to blast analysis
[8,21,40–42]. In contrast, the 9R-DOX-AOS subfamily is found in A.
terreus, A. niger, and a few other aspergilli [27,30,43]. The oxidation of
18:2n-6 in headfirst position by An-9R-DOX-AOS suggests a closer
catalytic relationship to 8R-DOX than can be predicted by sequence
homology with only 40% identity (cf. Fig. 2C). The 9S-DOX-AOS sub-
family also appears to be common in both ascomycetous and basidio-
mycetous fungi [16]. The evolution of the 9S-DOX-AOS and the 8R-
DOX-LDS subfamilies is interesting. At-8R-DOX-5,8-LDS oxidizes 18:2n-
6-Ile and 18:2n-6-Gly in tail first position mainly at C-9 and Fo-9S-DOX-
AOS oxidizes 18:2n-6 in this way. This could be a coincidence, but it
may also be an evolutionary trait.
An ancestor of 8R-DOX may have been modified in the evolutionary
process to bind fatty acids in reverse orientation to become a precursor
to 9S-DOX-AOS (Fig. 9B), but this process may also have followed other
paths. Evolution has nonetheless modified the active sites of 8R- and 9S-
DOX for presenting the fatty acids in optimal but opposite configuration
for catalysis. The LDS family is unique in this respect as the COX and α-
DOX families bind fatty acids in only one orientation [1,6,13]. The
ancestor to the peroxidase-COX superfamily is unknown. Its closest
relatives may be found in the bacterial “COX” family, which contains
fatty acid DOX [44].
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The orientation of fatty acids in the active sites of LDS subfamilies
must be determined experimentally and cannot presently be deduced
by sequence homology. 8R-DOX-LDS and An-9R-DOX-AOS bind 18:2n-6
headfirst in the active site, whereas Fo-9S-DOX-AOS binds 18:2n-6 tail
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Conflict of interest
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(2015) 1243–1252.
The author declares no conflict of interest.
Transparency document
The Transparency document associated with this article can be
found in the online version.
Acknowledgements
[23] W.L. Smith, W.E. Lands, Oxygenation of unsaturated fatty acids by soybean lipox-
ygenase, J. Biol. Chem. 247 (1972) 1038–1047.
Supported by Vetenskapsrådet Grant K2013-67X-06523-31-3, The
Knut and Alice Wallenberg Foundation Grant KAW 2004.0123, and by
Uppsala University. The generous gifts of deuterated linoleic acid by
Prof M. Hamberg, The Karolinska Institute, and the expression plasmid
for 9R-LOX by Dr. A. Brash, Vanderbilt University, are gratefully ac-
knowledged. I wish to thank Dr. I. Hoffmann for constructs for re-
combinant enzyme expression and Ms. Julia Swan for linguistic control
of the text.
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cyclooxygenase superfamily with amino acid derivatized polyenoic fatty acids,
Arch. Biochem. Biophys. 640 (2018) 93–101.
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