Oxygenation reactions catalyzed by the F557V mutant of soybean lipoxygenase-1: Evidence for two orientations of substrate binding

Article abstract of DOI:10.1016/j.abb.2019.108082

Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.

Full text of DOI:10.1016/j.abb.2019.108082

ArchivesofBiochemistryandBiophysics674(2019)108082
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Oxygenation reactions catalyzed by the F557V mutant of soybean
lipoxygenase-1: Evidence for two orientations of substrate binding
Dillon Hershelman, Kirsten M. Kahler, Morgan J. Price, Iris Lu, Yuhan Fu, Patricia A. Plumeri,
Fred Karaisz, Natasha F. Bassett, Peter M. Findeis, Charles H. Clapp∗
Department of Chemistry, Bucknell University, Lewisburg, PA, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Lipoxygenase
Mutagenesis
Regioselectivity
Stereoselectivity
Substrate binding
Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-
HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind
substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl.
Acad. Sci. USA 96 (1999) 4192–4197) have identified an important residue, corresponding to phe-557 in soy-
bean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of
linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of
13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-
first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which
leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced
33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12%
with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-
HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic
acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to
13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachi-
donic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and
ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be
altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence
that 13(S)-HPOD arises from tail-first binding.
1. Introduction
smaller beta barrel domain at the N-terminus. Mammalian lipox-
ygenases insert oxygen into different positions of arachidonic acid, and
Lipoxygenases catalyze the incorporation of molecular oxygen into
Z,Z-1,4 diene units in fatty acids and fatty acid derivatives to produce
conjugated diene hydroperoxides [1–3]. In animals, lipoxygenases in-
itiate the conversion of arachidonic acid into leukotrienes, lipoxins and
other signaling molecules [4,5]. In plants, lipoxygenases catalyze the
initial step in the conversion of 18-carbon polyunsaturated fatty acids
into jasmonic acid and other oxylipins [6]. Lipoxygenases are also
found in fungi and in some bacteria [6,7]. Some lipoxygenases will act
on the fatty acyl side chains in complex lipids [7–10], and these en-
zymes are involved in membrane modification [11], storage lipid mo-
bilization [12], ferroptosis [13], and skin barrier formation [14].
Most lipoxygenases are non-heme iron proteins, although some
utilize manganese in place of iron. The metal is bound in the center of a
large helical domain [15–19]. Most lipoxygenases also contain a
the molecular basis for this specificity is not readily apparent from the
crystal structures. Progress towards understanding the positional and
stereoselectivity of these enzymes has been reviewed [19]. A similar
challenge is presented by plant lipoxygenases, some of which in-
corporate oxygen at C-13 of linoleic acid whereas others add oxygen to
C-9 [3].
Studies on soybean lipoxygenase-1 (SBLO-1) have provided most of
our mechanistic understanding of the lipoxygenase reaction. This en-
zyme has a typical lipoxygenase structure, with the non-heme iron
bound in the interior of a C-terminal helical domain (Fig. 1) [15,16].
Wild-type SBLO-1 catalyzes the conversion of linoleic acid to primarily
13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD). At pH
7.5, about 10% of 9(S)-hydroperoxy-10E,12Z-octadecadienoic acid
(9(S)-HPOD) is also formed [20]. At pH 9.0, where the enzyme exhibits
∗ Corresponding author. Department of Chemistry, Bucknell University, One Dent Drive, Lewisburg, PA, 17837, USA.
E-mail address: cclapp@bucknell.edu (C.H. Clapp).
https://doi.org/10.1016/j.abb.2019.108082
Received 3 July 2019; Received in revised form 21 August 2019; Accepted 23 August 2019
Availableonline29August2019
0003-9861/©2019ElsevierInc.Allrightsreserved.

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
Abbreviations
8-HPETE 8-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid
8-HETE 8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid
14-HPND 14-hydroperoxy-10Z,12E-nonadecadienoic acid
14-HND 14-hydroxy-10Z,12E-nonadecadienoic acid
10-HPND 10-hydroperoxy-11E,13Z-nonadecadienoic acid
10-HND 10-hydroxy-11E,13Z-nonadecadienoic acid
11(S)-DLA 11(S)-deuteriolinoleic acid
GC/MS gas chromatography/mass spectrometry
HPLC
high pressure liquid chromatography
13-HPOD 13-hydroperoxy-9Z,11E-octadecadienoic acid
13-HOD 13-hydroxy-9Z,11E-octadecadienoic acid
KIE
kinetic isotope effect
9-HPOD 9-hydroperoxy-10E,12Z-octadecadienoic acid
9-HOD 9-hydroxy-10E,12Z-octadecadienoic acid
15-HPETE 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid
15-HETE 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid
11-HPETE 11-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid
11-HETE 11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid
10,13-NDA 10Z,13Z-nonadecadienoic acid
NMR
PLPC
nuclear magnetic resonance
1-palmitoyl-2-linoleoylphosphatidylcholine;
SBLO-1 soybean lipoxygenase-1
TCEP tris(2-carboxyethyl)phosphine hydrochloride.
Fig. 1. Structure of SBLO-1 (left); key residues and
two surface helices (right). The image at right is
rotated approximately 90° relative to the image at
left. Phe-557 is displayed in blue, arg-707 in red,
iron in yellow, and helix-2 in orange. In the image at
right, the green helix includes residues 536–546.
The figure was constructed from PDB file 3pzw
using PyMOL 2.3 (Schrodinger). (For interpretation
of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
maximal activity, the yield of 9-HPOD is reduced to 2–3% [20,21].
SBLO-1 will also oxygenate other fatty acids and substrates that have a
Z,Z-1,4-diene unit that begins on the ω6 position, and the major pro-
duct contains oxygen in the ω6 position [8,10,22].
accommodated by “tail-first” binding models, in which the terminal
methyl group is bound in a hydrophobic pocket in the interior of the
enzyme, the double bonds are near the iron, and the carboxy terminus
is near the surface (see Scheme 2A) [3,29,30]. Such a model provides
an explanation for the specificity for the ω6 position. It also accounts
for ability of the enzyme to oxygenate phospholipids [8,10], other
substrates with large head groups [31], and substrates with positively
charged head groups [32], since these groups could remain largely
outside the protein. In silico structures have been presented in which
linoleic acid binds in an internal cavity with the double bonds near the
iron and the methyl terminus near phe-557 [29,30] (displayed in blue
in Fig. 1). These models require some movement of the surface of the
protein to provide access to the internal cavity. Spin-labelling studies
suggest that the mobility of helix-2 (displayed in orange in Fig. 1) might
be important in providing access [33]. Studies with substrate analogues
with spin labels in the head group indicate that the head groups are
bound at the surface near helix-2 [34,35].
Studies in several laboratories have produced a body of evidence
[23–27] that supports the catalytic cycle for SBLO-1 presented in
Scheme 1. The initial step is the transfer of a hydrogen atom from the
pro-S position of the bisallylic carbon of the substrate to a ferric hy-
droxide species to produce a pentadienyl radical and the ferrous form of
the enzyme. The very large, nearly temperature-independent deuterium
kinetic isotope effect (KIE = 80) [26] on the reaction is attributed to
hydrogen tunneling [25–28]. Reaction of O2 with C-13 of the bound
pentadienyl radical leads to 13(S)-HPOD. An oxygen channel as a major
determinant of the observed stereo- and regiospecificity has been pro-
posed [21].
The manner in which substrates bind to SBLO-1 is uncertain. No
crystal structure of SBLO-1 with a substrate analogue bound at the
active site has been reported. The substrate specificity is most readily
An alternative possibility is that linoleic acid binds in “head-first”
Scheme 1. Catalytic cycle for the oxygenation of linoleic acid by SBLO-1.
2

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
Scheme 2. (A) Tail-first binding of linoleate to SBLO-1 and (B) head-first binding of linoleate to the F557V mutant according to the proposal by Hornung et al.29
.
manner, with the carboxylate group interacting with arg-707 (displayed
in red in Fig. 1) in the interior of the protein. Precedence for this
binding mode is provided by the crystal structure of 13(S)-HPOD bound
to soybean lipoxygenase-3 [36]. This enzyme is 80% identical to SBLO-
1, and all of the residues close to the bound 13(S)-HPOD are identical in
SBLO-1 and SBLO-3. The carboxy group of 13(S)-HPOD is bound close
to arg-726, which aligns with arg-707 in SBLO-1. Mutagenesis studies
on SBLO-1 suggest that arg-707 contributes to binding of linoleate [37],
and recent quantum mechanical studies on the mechanism of the li-
poxygenase reaction are based on head-first binding with the carbox-
ylate group of linoleate bound near arg-707 [27,38,39]. It is difficult to
reconcile head-first binding with the ability of SBLO-1 to oxygenate
substrates with large and positively charged head groups. Gardner has
proposed that the conversion of linoleic acid to 13(S)-HPOD by SBLO-1
occurs by tail-first binding, and that the 9(S)-HPOD that is formed is the
result of head-first binding [20]. Studies by Rickert and Klinman on
11(S)-deuteriolinoleic acid provide strong evidence that the formation
of 9(S)-HPOD results from binding in the opposite orientation as that
leading to 13(S)-HPOD [26].
Hornung et al. [29] have called attention to the importance of the
residue that corresponds to phe-557 in SBLO-1. These authors noted
that plant lipoxygenases with phenylalanine or histidine in this position
converted linoleic acid to predominantly 13(S)-HPOD, and lipox-
ygenases with valine in this position converted linoleate to primarily
9(S)-HPOD. The importance of this position was tested on cucumber
lipoxygenase, in which his-608 aligns with phe-557 in SBLO-1. The
wild-type cucumber enzyme converts linoleic acid to primarily (84%)
13(S)-HPOD. In contrast, the H608V mutant produces 95% 9(S)-HPOD.
Since the structure of cucumber lipoxygenase is unknown, Hornung
et al. used the known structure of SBLO-1 to propose an explanation for
these results. They proposed that bulky residues (phe or his) in position
557 (in SBLO-1) impede access to the conserved arg-707 in SBLO-1
(arg-758 in cucumber lipoxygenase) and thus favor formation of 13(S)-
HPOD (Scheme 2A), and that smaller residues in position 557 favor
9(S)-HPOD (Scheme 2B). The 9-HPOD formed by the H608V mutant of
cucumber lipoxygenase has the S-configuration at C-9, as expected for
head-first binding if O2 approaches the pentadienyl radical in the same
manner as in tail-first binding. Also consistent with the authors’ pro-
posal is the finding that trilinolein, a large substrate for which head-first
binding would be difficult, was oxygenated at C-13 by both wild-type
cucumber lipoxygenase and the H608V mutant [29].
evidence that oxygenation in the ω6 position results from tail-first
binding, and oxygenation at the ω10 position results from head-first
binding. This conclusion is also supported by experiments with the
F557V, R707L double mutant.
2. Materials and methods
2.1. Materials
Racemic 11-deuteriolinoleic acid was synthesized and en-
zymatically resolved by methods similar to those previously reported
[26,40]. The procedures are presented in the Supplementary Material.
Linoleic acid and 10Z,13Z-nonadecadienoic acid were obtained from
NuChek.
Arachidonic
acid
and
1-palmitoyl-2-linoleoylpho-
sphatidylcholine were obtained from Sigma Aldrich. 13(S)-HPOD was
prepared enzymatically from linoleic acid using wild-type SBLO-1 and
reduced with NaBH4 to give 13(S)-hydroxy-9Z,11E-octadecadienoic
acid (13(S)-HOD). 9(S)-HOD, ( )13-HOD, ( )9-HOD, 15(S)-HETE
and ( )11-HETE were obtained from Cayman Chemical.
Plasmid (pT7/L-1) [41] for the expression of SBLO-1 was provided
by Professor Ted Holman, University of California, Santa Cruz. Quick
Change Lightning kits (Agilent) were used for site-directed mutagen-
esis. Mutant plasmids were isolated using Qiagen Mini Prep kits, and
the entire lipoxygenase coding sequence in each mutant plasmid was
sequenced by the Penn State Genomics Core Facility, University Park,
PA.
2.2. Enzyme expression and purification
Expression was carried out by a modification of a published pro-
cedure [41]. The wild-type and mutant enzymes were purified by
chromatography on SP-Sephadex [42] at pH 6.0 followed by chroma-
tography on DEAE Sephadex [43] at pH 6.8 to yield protein that
was > 90% pure by SDS-PAGE. Experimental details are presented in
the Supplementary Material. Concentrations of purified enzyme were
0.1%
determined spectrophotometrically using A
= 1.6 [44].
280
2.3. Kinetic assays
Formation of the conjugated diene in the products was monitored
spectrophotometrically at 234 nm (ε234 = 2.5 × 104 M−1 cm−1
at
)
In the present work, we have tested the proposal of Hornung et al.
by studying the F557V mutant of SBLO-1. The mutation shifts the
product distribution in favor of 9(S)-HPOD, but not by enough to make
this the major product. Experiments with 11(S)-deuteriolinoleic acid
indicate that 9(S)-HPOD formed by the mutant results from loss of the
pro-R hydrogen from C-11, as expected for binding in the opposite or-
ientation from that leading to 13(S)-HPOD (Scheme 2). Experiments
with 10,13-nonadecadienoic acid and arachidonic acid provide
25 °C using a Beckman DU 640 spectrophotometer. Substrate solutions
were prepared in 50 mM sodium borate, pH 9.0, 25 °C. Reactions were
initiated by addition of enzyme, and the initial rates were determined
from the first 10% of the reaction. Michaelis-Menten parameters were
obtained by nonlinear regression using Sigma Plot. Routine activity
measurements were carried out at 72 μM linoleic acid. In the case of the
F557V,R707L double mutant, 2.5 μM 13(S)-HPOD was also present to
reduce the induction period observed with this mutant. One unit of
3

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
activity corresponds to formation of diene hydroperoxide at a rate of
1.0 μmol/min.
trimethylsilyldiazomethane [46]. In the latter method, solutions of
about 150 μg of fatty acid in 1.0 mL of methanol/toluene (2:1) were
treated with 30 μL of 2.0 M trimethylsilyldiazomethane in hexanes
(Sigma Aldrich). After incubation for 2 h at room temperature, the
solvents and reagents were removed by evaporation under nitrogen,
and the residue was dissolved in 300 μL of hexane for analysis by HPLC
or GC/MS.
2.4. Reaction of fatty acid substrates with SBLO-1 and mutants for product
analysis
A 40-μg (0.43 nmol) aliquot of wild-type or mutant SBLO-1 was
added to 50 mL of a magnetically stirred solution of linoleic acid
(15–200 μΜ) in 100 mM tris buffer, pH 7.5, or 50 mM sodium borate
buffer, pH 9.0, at room temperature. The progress of the reaction was
monitored by periodically measuring A234. When the absorbance le-
veled off, 29 mg of TCEP was added to reduce hydroperoxides to the
corresponding alcohols, and the mixture was stirred at room tempera-
ture for 30 min. The solution was acidified to pH 3–3.5 with 1.0 M citric
acid and then extracted once with 30 mL of diethyl ether and twice with
15 mL of diethyl ether. The combined ether extracts were concentrated
by rotary evaporation, and the residue was dissolved in 1.0 mL of
hexanes for analysis by normal-phase HPLC.
Experiments with 11(S)-deuteriolinoleic acid were carried out in the
same manner, except that the reaction volume was 10 mL, the enzyme
concentration was 60 nM, and the extractions were carried out with
three 6-mL portions of diethyl ether. Experiments to determine the
product ratios with 10Z,13Z-nonadecadienoic acid (10,13-NDA) and
arachidonic acid were carried out as described for linoleic acid. To
identify the products from 10,13-NDA, the reaction was carried out by
incubating 8.6 nM of the F557V mutant with 100 mL of 200 μM 10,13-
NDA in 0.10 M tris, pH 7.5. The procedure was the same as in the
smaller-scale reactions, except that 58 mg of TCEP was used and the
volumes of diethyl ether used in the three extractions were doubled.
2.7. Oxygenation of 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC)
and transesterification of the products
A 100-μL aliquot of 10 mM PLPC in ethanol was added to a round-
bottomed flask, and the ethanol was evaporated under a stream of ni-
trogen. The residue was dissolved in 1.0 mL of 0.20 M sodium borate,
pH 9.0, containing 10 mM deoxycholate. Wild-type SBLO-1 or the
F557V mutant was added to a final concentration of 420 nM, and the
reaction mixture was stirred magnetically at room temperature.
Periodically, 15-μL aliquots were withdrawn and diluted with 585 μL of
buffer for A234 measurements. When A234 leveled off, TCEP (29 mg) was
added to the reaction mixture. After 30 min of stirring at room tem-
perature, the mixture was transferred to a 15-mL centrifuge tube and
extracted with 3 mL of chloroform/methanol (2:1) followed by 2 mL of
chloroform. The two extracts were combined and concentrated. The
residue was dissolved in 0.5 mL of methanol, and the resulting solution
was concentrated under nitrogen to about 100 μL. Half of this solution
was mixed with 50 μL of 0.5 M sodium methoxide in methanol (Sigma
Aldrich) to convert the acyl side chains of the products to their methyl
esters. After 10 min at room temperature, the reaction was quenched by
addition of 325 μL of 0.1 M HCl, and the mixture was extracted with
two 325-μL portions of hexanes. The combined hexanes extracts were
washed with 325 μL of water and concentrated to about 0.5 mL for
analysis by HPLC.
2.5. Analytical methods
Normal-phase HPLC was carried out using
a 250 × 4.6 mm
Adsorbosphere Silica 5μ column (Alltech) or a 250 × 4.6 mm HAISIL
Silica 5μ column (Higgins Analytical) eluted isocratically at 1.0 mL/
min. In most experiments, the mobile phase was hexanes/isopropanol/
acetic acid (97.5:2.5:0.1). When other mobile phases were used, these
are indicated in the Results section. UV detection of diene hydroper-
oxides was carried out at 234 nm. Chiral-phase HPLC was performed on
a 250 × 4.6 mm Daicel Chiralpak AD-H column eluted isocratically
with hexanes/ethanol (95:5) at 1.0 mL/min. GC/MS analyses were
3. Results
3.1. Oxygenation of linoleic acid by the F557V, F557A and F557S mutants
Reactions were carried out at room temperature, and the relative
amounts of 13-HPOD and 9-HPOD were determined by reduction of the
hydroperoxide products to the corresponding alcohols (13-HOD and 9-
HOD), which were analyzed by HPLC. The results are summarized in
Table 1.
carried out on
a
Hewlett/Packard GCD instrument with
a
12 m × 0.3 mm HPI capillary column. The column temperature was
maintained at 50 °C for 3 min and then increased from 50 to 250 °C at
20°/min.
At pH 9.0, oxygenation of linoleic acid (50 μM) by the F557V mu-
tant produced 13-HPOD and 9-HPOD in a ratio of 83:17, compared with
Electrospray mass spectrometry was carried out on a Thermo
Fischer Scientific Exactive Mass Spectrometer (version 1.1). Samples
(2.0 μL) of 13-HOD or 9-HOD (15–95 μM) in methanol were analyzed
by flow injection at 30 μL/min of methanol delivered by an Accela 1250
pump and injected through a Rheodyne automated 6-way injection
valve (PD715-000). The ionization source was an electrospray ionizer
alternating between 3 kV and −2.5 kV, and the mass analyzer was an
orbitrap recording fragments in the 50–800 m/z range. The typical re-
solution of the analyses was greater than 45,000. The software used to
record the analyses was Thermo Fischer Scientific Xcalibur™ (version
3.0). Raw intensity values used for deuterium content calculations were
from negative-ion mode scans. The method for calculating the percent
deuteration from the peak intensities is described in the Supplementary
Material.
Table 1
Regioselectivity of the oxygenation of linoleic acid by wild-type SBLO-1 and
site-directed mutants.
Enzyme
pH
Linoleic acid (μM)
% 13a
%9a
n
Wild type
9.0
9.0
7.5
9.0
9.0
7.5
7.5
7.5
7.5
9.0
7.5
7.5
7.5
9.0
50
200
50
97
94
92
83
81
61
63
65
59
96
88
91
88
95
3
6
8
1
1
2
1
1
2
5
1
1
1
3
2
3
1
1
1
F557V
50
17
19
39
37
35
41
4
12
9
12
5
200
200
50
30
15
50
50
50
50
1
3
1
3
F557A
3
1
2
3
2
F557S
F557V, R707L
1
2.6. Derivatizations
50
a
Catalytic hydrogenation and the preparation of trimethylsilyl deri-
vatives were carried out as described previously [45]. Conversion of
fatty acid products to methyl esters was carried out using diazo-
Determined by HPLC from the relative intensities of the peaks for 13-HOD
and 9-HOD obtained by reduction of 13-HPOD and 9-HPOD formed in the
enzymatic reaction. In some cases, the reported value is the mean of n replicate
methane,
as
described
previously
[45],
or
with
experiments
standard deviation (n ≥ 3) or
average deviation (n = 2).
4

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
97:3 for the wild-type enzyme. At pH 7.5, the ratio of 13-HPOD to 9-
HPOD was 63:37 for the F557V mutant compared with 92:8 for the
wild-type. The ratios observed with the wild-type enzyme are in rea-
sonable agreement with previously reported values [20,21]. The results
with the F557V mutant at pH 7.5 were obtained with three different
preparations of this mutant. The results indicate that replacing phe-557
with valine increases the yield of 9-HPOD, but not by a large enough
amount to make it the major product.
To analyze the stereochemistry, 13-HOD and 9-HOD from an ex-
periment with the F557V mutant at pH 7.5 were converted to their
methyl esters, which were purified by normal-phase HPLC and analyzed
by chiral-phase HPLC (Fig. S2). The configuration at C-13 of methyl 13-
HOD was primarily S (S:R = 95:5), as is the case with wild-type SBLO-
It was found that the activity of the F557V,R707L mutant in crude
lysates could be increased to about 10% of wild-type level if the ex-
pression procedure was altered to increase the amount of time the cells
were incubated with ethanol prior to harvesting [47] (see Supplemental
Data). This modified procedure also appeared to increase the stability of
the activity. Analysis of the products formed from linoleic acid by the
crude lysate at pH 7.5 gave the same product ratio (13-HOD/9-
HOD = 89:11) as obtained with our standard expression procedure.
The F557V,R707L mutant expressed by the modified procedure was
purified to yield homogeneous enzyme with a specific activity of 15
units/mg, in contrast to values of 140–180 units/mg that we typically
obtain with wild-type enzyme. Product analyses were carried out by the
same procedures used to obtain the data in Table 1. Three experiments
at pH 7.5 gave an average 13-HOD/9-HOD ratio of 88( 2):12( 2).
The chromatograms also contain two minor unidentified products, each
corresponding to 1–2% of the total (see Fig. S3),. These products were
also observed in the experiments with crude lysates. At pH 9.0, the 13-
HOD/9-HOD ratio was 95:5, and the minor products were not observed.
Comparison of these results with those for the F557V mutant indicates
that replacing arg-707 with leucine shifts the product ratio in favor of
oxygenation at C-13, as expected from the model in Scheme 2.
The 13-HOD and 9-HOD from one of the experiments with the
purified mutant at pH 7.5 were converted to their methyl esters, which
were purified by HPLC. Analysis by chiral-phase HPLC indicated the
methyl 13-HOD was almost entirely S (S/R = 98:2). The methyl 9-HOD
was also predominately S, but the enantioselectivity (S/R = 77:23) was
lower than that obtained with the F557V mutant.
1. The configuration of 9-HOD at C-9 was also primarily
S
(S:R = 87:13). The 9(S) enantiomer is expected if the 9-oxygenated
product results from binding in the opposite orientation from the one
leading to 13-oxygenation, assuming O2 approaches C-9 of the bound
pentadienyl radical through the same channel that is used in oxyge-
nation at C-13.
Kinetics experiments on the oxygenation of linoleic acid by the
F557V mutant were carried out at pH 9.0 using a spectrophotometric
assay that measures the total rate of diene hydroperoxide formation.
The kinetics give a reasonable fit to the Michaelis-Menten equation at
substrate concentrations up to 40 μM with kcat = 76
m = 13 1 μM. Studies in our lab with wild-type SBLO-1 give kcat
values in the range of 230–270 s−1 and Km = 13
1 μM. The lower
4 s−1 and
K
kcat of the F557V mutant suggests that phe-557 may be important for
optimal positioning of linoleic acid for C(11)-H abstraction. The F557V
mutant exhibits substrate inhibition at concentrations above 60 μM.
The data in Table 1 indicate that the ratio of 13-HPOD to 9-HPOD does
not vary appreciably from 15 to 200 μM linoleic acid. These results
indicate that although high concentrations of substrate reduce the rate
of the reaction, they do not substantially affect the product ratio.
Following the proposal of Hornung et al. [29], our working hy-
pothesis was that the smaller size of valine compared with phenylala-
nine is responsible for the change in product ratio observed with the
F557V mutant. This reasoning suggests that the product ratio might be
further shifted towards 9-HPOD by introducing residues at position 557
that are smaller than valine. Contrary to this expectation, the F557A
mutant gave a 13-HPOD/9-HPOD ratio of 88:12 at pH 7.5 and 96:4 at
pH 9. The 13-isomer had an S/R ratio of 98:2. The F557S mutant gave a
ratio of 91:9 at pH 7.5. Clearly, the size of the residue at position 557 is
not, by itself, a reliable predictor of the product ratio.
3.3. Oxygenation of 11(S)-Deuteriolinoleic acid by the F557V mutant and
wild-type SBLO-1
The formation of 13(S)-HPOD by wild-type SBLO-1 is initiated by
removal of hydrogen from the pro-S position [22,48] on C-11 of linoleic
acid (Scheme 2A). If formation of 9(S)-HPOD by the F557V mutant
occurs by binding in the opposite orientation, this process would be
expected to involve removal of hydrogen from the pro-R position
(Scheme 2B). To test this prediction, we have investigated the action of
F557V on 11(S)-deuteriolinoleic acid (11(S)-DLA). This material was
prepared by a method used previously by Rickert and Klinman [26] and
by Hamberg [40]. Racemic 11-deuteriolinoleic acid was chemically
synthesized and kinetically resolved by incubation with a low con-
centration (3 nM) of wild-type SBLO-1. Because of the specificity of
SBLO-1 for the pro-S position of C-11 and the very large kinetic isotope
effect, the enzyme preferentially oxygenates the 11(R) enantiomer.
After about 50% conversion, the rate of product formation (as judged
by A234) decreased markedly. After 60% conversion, the reaction was
quenched by acidification, and the unreacted 11-deuteriolinoleic acid
was extracted and purified by HPLC. This material should be highly
enriched with the 11(S) enantiomer.
3.2. Oxygenation of linoleic acid by the F557V,R707L double mutant
According to the proposal of Hornung et al. [29], the formation of
9(S)-HPOD from linoleic acid by the F557V mutant involves interaction
of the carboxylate group of the substrate with arg-707 in SBLO-1. To
test this hypothesis, we decided to investigate the F557V,R707L double
mutant. A possible complication with this experiment is that arg-707 is
a conserved residue that forms a hydrogen bond with asp-490 in the
interior of the protein, and loss of this interaction might impede folding
and/or alter the three-dimensional structure. Hornung et al. prepared
the corresponding double mutant (H608V,R758L) of the cucumber
enzyme and found that it had only 5% of the wild-type activity and
converted linoleic acid to a 60:40 mixture of 13-HPOD and 9-HPOD,
both of which were racemic mixtures. We interpret these results as
evidence that the pentadienyl radical intermediate dissociated from the
enzyme and reacted with O2 in solution.
When the F557V,R707L double mutant of SBLO-1 was expressed
according to our standard protocol, the lipoxygenase activity in crude
cell lysates was only about 6% of what is customarily obtained with
wild type. A sample of crude lysate was incubated with 50 mM linoleic
acid at pH 7.5, and the products were reduced with TCEP and analyzed
by HPLC. The ratio of 13-HOD to 9-HOD was 89:11.
Solutions of 11(S)-deuteriolinoleic acid (50 μM) in 100 mM tris
buffer, pH 7.5, were incubated at room temperature with 60 nM of the
F557V mutant, and the products were reduced by TCEP and analyzed
by HPLC, as in the experiments in Table 1. The results of three ex-
periments gave an average of 93.8
0.3% of 9-HOD, compared with
only 37% with unlabeled linoleic acid. The shift in the product ratio is
consistent with the hypothesis that oxygenation at C-9 requires removal
of hydrogen from the pro-R position of C-11 of linoleic acid. The for-
mation of 13(S)-HPOD is known [48] to involve loss of the pro-S hy-
drogen from C-11, and the rate of this process with 11(S)-DLA is ex-
pected to be greatly reduced by the large KIE. If 9-oxygenation requires
loss of hydrogen from the pro-R position, its rate should be reduced
only slightly with 11(S)-DLA, owing to the small secondary isotope
effect [26], so that the ratio of 9-oxygenation to 13-oxygenation should
increase, as is observed.
The 9-HOD and 13-HOD produced by oxygenation of 11(S)-DLA
followed by reduction were purified by HPLC and analyzed by negative-
5

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
ion electrospray mass spectrometry. The results of these experiments
are presented in Table 2, along with data on undeuterated standards of
9-HOD and 13-HOD. The spectrum of the 9-HOD standard has a pro-
minent M − 1 peak at m/z 295.2 and peaks at m/z 296.2 and 297.2
owing to naturally occurring isotopes. In the spectrum of 9-HOD ob-
tained from experiments with F557V and 11(S)-DLA, the signal at 295.2
is barely detectable, indicating that virtually all of the 9-HOD contains
deuterium. This is the result that is expected if oxygenation at C-9 in-
volved loss of hydrogen from the pro-R position at C-11. Quantitative
analysis of the data from three experiments indicate that the percent of
bind normally. Consequently, if 14-HPND results from tail-first binding
and 10-NPND from head-first binding, the ratio of 14-NPND to 10-
NPND formed by the F557V mutant would be expected to be higher
than the ratio of 13-HPOD to 9-HPOD produced by the F557V mutant
from linoleic acid.
A 50-mL solution of 50 μM 10,13-NDA was incubated with 8.6 nM of
the F557V mutant in 0.100 M tris, pH 7.5. Treatment with TCEP fol-
lowed by extraction and analysis by HPLC (Fig. 2) revealed a major
product (peak 1) and three minor products (peaks 2–4). The same
products were formed when the reaction was carried out with wild-type
SBLO-1, but peak 3 was smaller (Table 4).
deuteration is 99.4
0.4%. The 1H NMR spectrum of this product
(Fig. S4) is consistent with the presence of deuterium at C-11.
Conversion of the deuterated 9-HOD to its methyl ester followed by
chiral-phase HPLC indicated that the configuration at C-9 is almost
entirely S (S:R = 98:2). Combined with the specificity for the pro-R
position at C-11, this result indicates that the overall stereochemical
course of the reaction is antarafacial, which is characteristic of most
lipoxygenase reactions and consistent with the working hypothesis in
Scheme 2B.
To identify the products, the reaction was carried out on a larger
scale (100 mL of 200 μM 10,13-NDA), and the products were reduced
with TCEP, extracted, and purified by HPLC. The major product (peak
1) gave an 1H NMR spectrum (Fig. S5) that showed the characteristic
resonances for a E,Z-diene with a hydroxyl group on a carbon next to
the E double bond. The spectrum is consistent with either 14-hydroxy-
10Z,12E-nonadecadienoic acid (14-HND) or 10-hydroxy-11E,13Z-non-
adecadienoic acid (10-HND). To distinguish between these regioi-
somers, the product was methylated with diazomethane, trimethylsi-
lylated and catalytically hydrogenated. The electron-impact mass
spectrum of the resulting derivative (Fig. S6A) exhibited fragment ions
at m/z 173 [CH3(CH2)4CHOSi(CH3)3] and m/z 329 [(CH3)3SiOCH
(CH2)12CO2CH3], which indicate that the trimethylsiloxy group is at-
tached to C-14. These results establish that the major product of the
enzymatic reaction is 14-HPND. Peak 3 was also purified and deriva-
tized in the same manner. The electron-impact mass spectrum (Fig.
S6B) of this derivative gave ions at m/z 229 [CH3(CH2)8CHOSi(CH3)3]
and m/z 273 [(CH3)3SiOCH(CH2)8CO2CH3], which indicates that the
trimethylsiloxy group is on C-10 and establishes that peak 3 arises from
oxygenation at C-10. This product is most likely 10-HND, but we did
not obtain enough of this substance to confirm the stereochemistry of
the diene by NMR. Peaks 2 and 4 were not identified; they may be E,E
isomers (see Section 3.5).
In the mass spectrum of the 13-HOD obtained from 11(S)-DLA
(Table 2), the peak at 296.2 is more intense than the peak at 295.2,
indicating the presence of a large amount of deuterium. Quantitative
analysis indicates that the 13-HOD is 52.0
1.4% deuterated. This
result was initially surprising, since if oxygenation at C-13 involves
removal of deuterium from the pro-S position at C-11, the 13-HOD
would be expected to contain no deuterium. The product ratios and the
percentages of deuterium in each regioisomer were used to calculate
the overall distribution of deuterated and undeuterated products
(Table 3). The amount of deuterated 13-HOD that is formed is only
about 3% of the total product mixture. This material might arise from
loss of hydrogen from a small amount of residual 11(R)-DLA in our
sample. Alternatively, it could arise by loss of hydrogen from 11(S)-
DLA, if the large KIE for deuterium removal caused the enzyme to re-
move hydrogen from the pro-R position of 11(S)-DLA in spite of its
normal specificity for the pro-S position. Rickert and Klinman have
suggested a similar loss of stereochemical fidelity with wild-type SBLO-
1 at high pH [26]. In our case, it is not possible to distinguish between
these two possible explanations for the presence of deuterated 13-HOD.
The oxygenation of 11(S)-DLA with wild-type SBLO-1 was carried
out at pH 7.5 under the same conditions as those used for the F557V
The relative intensities of peaks 1–4 obtained with the F557V mu-
tant indicate that 14-HPND amounts to 85% of the product mixture,
compared with 63% of 13-HPOD produced from linoleic acid under the
same conditions. Addition of one methylene group to the carboxy end
of the substrate clearly favors formation of the product that is oxyge-
nated at the ω6 position at the expense of the product oxygenated at the
ω10 position. This result is consistent with the hypothesis that ω6
oxygenation results from tail-first binding and ω10 oxygenation results
from head-first binding.
mutant. Three experiments gave an average of 78
8% of the 9-
oxygenated product compared with 8% of this regioisomer with un-
deuterated linoleic acid. Electrospray MS analysis indicated that the 9-
oxygenated product was 99.6
nated product was 11 3% deuterated. The overall distribution of
0.1% deuterated and the 13-oxyge-
Oxygenation of arachidonic acid (50 μM) by the F557V mutant was
carried out at pH 7.5 followed by treatment with TCEP. HPLC analysis
of the product mixture showed two peaks in a ratio of 92:8, which had
the same retention times as 15-HETE and 11-HETE, respectively. GC/
MS of the material from the major peak (after methylation, catalytic
hydrogenation and trimethylsilylation) confirmed that it is 15-HETE
(Fig. S7A). The same method applied to the material in the minor peak
indicates the presence of both 11-HETE and 8-HETE (Fig. S7B). A
control experiment with the wild-type enzyme gave only 15-HETE. The
strong preponderance of 15-oxygenation by the F557V mutant is con-
sistent with the hypothesis that 15-oxygenation arises from tail-first
binding and the minor products from head-first binding.
deuterated and undeuterated products is presented in Table 3. These
results are similar to those obtained by Rickert and Klinman, who found
that oxygenation of 11(S)-DLA by wild-type SBLO-1 at pH 8.0 gave 80%
9-HPOD [26]. Our results at pH 7.5 indicate that the amount of 9-HPOD
produced by the wild-type enzyme is less than the amount of 9-HPOD
produced by the F557V mutant under the same conditions. This result is
consistent with the generalization that replacing phe-557 with valine
increases oxygenation at C-9.
3.4. Oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid
by the F557V mutant
Table 2
Compared with linoleic acid, 10Z,13Z-nonadecadienoic acid (10,13-
NDA) contains one additional methylene group between the diene unit
and the carboxy terminus. Oxygenation of this substance by wild-type
or mutant SBLO-1 could give either 14-hydroperoxy-10Z,12E-non-
adecadienoic acid (14-HPND) or 10-hydroperoxy-11E,13Z-non-
adecadienoic acid (10-HPND, Scheme 3). If 10,13-NDA bound in a
head-first manner, the presence of the extra methylene group might
force the diene unit to be poorly positioned for hydrogen abstraction
from C-12. In contrast, tail-first binding should allow the diene unit to
Relative intensities of signals in the ESI mass spectra of 13-HOD and 13-HOD
standards and samples from reduction of products of the oxygenation of 11(S)-
DLA by the F557V mutant.
m/z
295.2
296.2
297.2
298.2
9-HOD (standard)
9-HOD (from 11(S)DLA)
13-HOD (standard)
100.0
0.40
100.0
73.94
20.08
100.0
19.81
100.0
1.61
21.88
1.67
< 0.1
1.41
< 0.1
1.15
13-HOD (from 11(S)DLA)
18.22
6

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
Table 3
mutant was also carried out at pH 7.5, and the products were reduced
and methylated as described above. The resulting HPLC chromatograms
were more complex and variable than those obtained at pH 9.0. In
addition to higher intensities for peaks 2–4, there were additional
unidentified peaks. These observations suggest that dissociation of the
pentadienyl radical is more prevalent at pH 7.5 than at pH 9.0 for both
wild-type and mutant.
Percentages of deuterated and undeuterated products obtained by oxygenation
of 11(S)-DLA by wild-type SBLO-1 and the F557V mutant.
Enzyme
Percentages of Total Products
Deuterated 13-
HOD
Unlabeled 13-
HOD
Deuterated 9-
HOD
Unlabeled 9-
HOD
F557V
3.25 ( 0.14)
3.00 ( 0.14)
20 ( 7)
93.2 ( 0.5)
78 ( 8)
0.6 ( 0.4)
0.35 ( 0.12)
Wild type 2.51 ( 1.2)
4. Discussion
The reported values are mean
standard deviation for three experiments.
Our results are consistent with the prediction of Hornung et al. that
replacement of phe-557 with valine should shift the product ratio in
favor of 9(S)-HPOD. However, the shift is not large enough to make
9(S)-HPOD the major product. This result is consistent with evidence
from other enzymes that although the identity of this residue is im-
portant, it is not the sole determinant of positional specificity in plant
and fungal lipoxygenases. For example, a lipoxygenase involved in sex
determination in maize, which was predicted to be a 13-lipoxygenase
and has a phenylalanine in this position, actually produces a 1:1 mix-
ture of 13-HPOD and 9-HPOD [51]. A 9-lipoxygenase in Solanum tu-
berosum has a valine in the position that aligns with phe-557 in SBLO-1,
but replacement of this residue with phenylalanine did not convert the
enzyme into a 13-lipoxygenase [52]. In spite of these exceptions, this
residue clearly has a strong influence on regioselectivity [3], and the
work reported here provides additional evidence for its importance. As
previously noted by Hornung et al. [29], phe-557 in SBLO-1 aligns with
met-419 in human ALOX15 [53], which has been shown to be an im-
portant specificity determinant for human ALOX15 and for ALOX15
homologs [54,55].
Most of our studies were carried out at 50 μM linoleic acid, pH 7.5,
where some aggregation of the linoleic acid probably occurs [56,57].
The absence of an appreciable effect of the linoleic acid concentration
on the product ratio (Table 1) suggests that the product ratio is not
strongly affected by the size of the aggregates.
Replacing phe-557 with a residue smaller than valine might be ex-
pected to cause a larger shift towards the 9-oxygenation. Contrary to
this expectation, the F557A and F557S mutants give only slightly more
9-oxygenation than wild type. It is possible that these smaller residues
allow the interior end of the binding pocket to collapse, making head-
first binding more difficult. To our knowledge, no lipoxygenases have
been reported that have serine or alanine in these positions.
Hornung et al. [29] proposed that 9(S)-HPOD arises from a binding
mode in which the carboxylate group of linoleate interacts with the
guanidino group of arg-707. Support for this proposal is provided by
our finding that the percentage of 9-HPOD produced from linoleic acid
is lower for the F557V,R707L double mutant than it is for the F557V
mutant. The 9-HPOD that is formed by the F557V,R707L mutant has S/
R = 77:23. The presence of substantial amounts of the R enantiomer
suggests that some of the product is formed by dissociation of the
pentadienyl radical intermediate followed by reaction with O2 in so-
lution. The enantiomeric ratio is consistent with 46% of the 9-HPOD
being a racemic mixture formed in solution and the remaining 54%
formed on the enzyme with the S configuration, as expected from
binding in the opposite orientation as the one leading to 13(S)-HPOD.
These results imply that head-first binding is still possible with the
F557V,R707L double mutant but is considerably less favorable than it is
with the F557V mutant.
3.5. Oxygenation of 1-palmitoyl-2-linoleoylphosphatidylcholine
SBLO-1 will oxygenate phospholipids that have a side chain with a
1Z, 4Z diene unit that begins on the sixth carbon from the alkyl ter-
minus. Because of the large head group, head-first binding by phos-
pholipid substrates is expected to be difficult or impossible. With this in
mind, we investigated the action of the F557V mutant on 1-palmitoyl-2-
linoleoylphosphatidylcholine (PLPC). Brash and coworkers [8] have
previously shown that the linoleoyl side chain of this substrate is oxy-
genated by wild-type SBLO-1 to the 13(S)-9Z,11E-octadecadienoyl de-
rivative at pH 9.0. The goal of our experiments was to determine
whether the F557V mutant would also be highly specific for oxygena-
tion at C-13.
When PLPC was incubated with the F557V mutant at pH 9.0, in the
presence of 10 mM deoxycholate, formation of conjugated diene could
be detected by an increase in absorbance at 234 nm. When the rise in
absorbance was complete, the products were treated with TCEP.
Subsequent treatment with sodium methoxide in methanol converted
the acyl side chains to their methyl esters, which were analyzed by
normal-phase HPLC. The resulting chromatogram (Fig. 3) showed a
major peak (peak 1) and three minor peaks (2–4). The relative in-
tensities of these peaks are given in Table 5. The same peaks were
obtained in experiments with wild-type SBLO-1, but in this case, the
relative intensity of peak 1 was much higher than with F557V (see
Table 5).
By comparison of the retention times with those of standards, peak 1
was identified as the methyl ester of 13-HOD, and peak 3 as the methyl
ester of 9-HOD. To identify peaks 2 and 4, a sample of methyl linoleate
was subjected to nonenzymatic autoxidation in the presence of N-hy-
droxyphthalimide, which has been shown to produce both the E,Z and
E,E isomers of the 13- and 9-hydroperoxides [49]. Following reduction
by triphenylphosphine, analysis by HPLC showed peaks 1–4 with si-
milar intensities for each peak (Table 5). This result implies that peaks 2
and 4 are E,E isomers.
The results in the preceding paragraph indicate that oxygenation of
the linoleoyl side chain of PLPC by the wild-type enzyme occurs almost
entirely on C-13, in agreement with the work of Brash [8]. The reaction
with the F557V mutant is more complex. The methyl 13-HOD (peak 1)
and methyl 9-HOD (peak 3) obtained in the experiment with the F557V
mutant were purified by HPLC and analyzed by chiral-phase HPLC. The
methyl 13-HOD gave a ratio of S/R of 89:11. In contrast, the methyl 9-
HOD gave an S/R ratio of 51:49. The finding that the 9-oxygenation
product is virtually racemic implies that it was formed in solution ra-
ther than at the active site. This could occur if the initially-formed
pentadienyl radical dissociated from the enzyme and reacted with
oxygen in solution. If the addition of oxygen is reversible, this process
would also lead to the formation of E,E-isomers [50].
The activity of the F557V,R707L double mutant is only about 10%
of the activity of the wild-type enzyme. Low activity could result from
the loss of the interaction between arg-707 and asp-490, but previous
Oxygenation of PLPC by both wild-type SBLO-1 and the F557V
Scheme 3. Hydroperoxides and alcohols derived
from 10,13-NDA.
7

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
Fig. 2. Normal-phase HPLC chromatogram of the products obtained by oxygenation of 10,13-NDA by the F557V mutant followed by reduction. The mobile phase
was hexanes/isopropanol/acetic acid (97.5:2.5:0.1).
Table 4
Table 5
Relative intensities of the HPLC peaks from the oxygenation of 10,13 NDA by
SBLO-1 and the F557V mutant at pH 7.5.
Relative intensities of the HPLC peaks from methanolysis of the reduced pro-
ducts of oxygenation of PLPC by wild-type SBLO-1 and the F557V mutant. The
numbers of the peaks correspond to those in Fig. 3. Also shown are the relative
intensities of the peaks for the reduced products of nonenzymatic autoxidation
of methyl linoleate.
Enzyme
Intensities (%)
1
2
3
4
Enzyme
pH
1
2
3
4
F557V
Wild type
85.7
90.5
1.2
3.5
3.7
4.0
1.4
1.1
7.6
2.1
0.4
0.9
2.9
3.4
0.2
2.4
Wild type
F557Va
Autoxidation
9.0
9.0
–
90
58
26
2
9
25
2
15
23
6
18
26
1
1
1
1
Peaks 1–4 refer to the labeled peaks in Fig. 2. Reported values are the
mean standard deviation for four experiments (F557V) or three experiments
(wild type).
a
Each reported value is the mean
average deviation from two replicate
experiments.
work showed that the R707L mutant of SBLO-1 had a kcat/Km value that
was 42% of wild type [37]. In light of this result, the low activity of the
double mutant suggests that the loss of the arg-707/asp-490 interaction
combined with the F557V mutation leads to low activity, presumably
by changes in conformation. These changes likely contribute to the
increased tendency of the pentadienyl radical to dissociate. In the case
of cucumber lipoxygenase [29], the corresponding double mutant
(H608V,R758L) appears to have an even higher rate of dissociation,
since the 13-HPOD and 9-HPOD produced from linoleic acid are ra-
cemic and formed in nearly equal amounts. These results provide no
information about substrate binding. Fortunately, the lower rate of
dissociation with the F557V,R707L mutant of SBLO-1 provides a high
enough level of regio- and stereoselectivity to provide evidence that
replacement of arg-707 with a nonpolar residue increases tail-first
binding at the expense of head-first binding. This conserved arginine
has been proposed to be generally important in head-first binding by
plant lipoxygenases that convert linoleate to 9(S)-HPOD [3]. To our
knowledge, the results reported here are the first case of a mutagenesis
experiment that provides support for this idea.
The experiments with 11(S)-DLA provide information about the
stereoselectivity of hydrogen abstraction from C-11, which was not
investigated in the work of Hornung et al. [29]. The isotope effect on
the product ratio that is observed with 11(S)-DLA and the high levels of
deuterium in the 9-oxygenated product indicate that oxygenation of
linoleic acid at C-9 catalyzed by the F557V mutant occurs with loss of
hydrogen from the pro-R position. Since oxygenation at C-13 results
from loss of hydrogen from the pro-S position [22,48], the
Fig. 3. Normal-phase HPLC chromatogram of the products obtained by oxygenation of PLPC by the F557V mutant followed by reduction and methanolysis. The
mobile phase was hexanes/isopropanol (98:2).
8

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
stereochemical results strongly imply that oxygenations at C-9 and C-13
result from different binding orientations. We have described these two
orientations as “opposite”, which is consistent with the stereochemistry,
but it is important to note that the stereochemical results do not require
that the orientations be exactly opposite. For example, in Scheme 2B,
the carboxylate group of linoleate does not occupy exactly the same
location that is occupied by the methyl group in Scheme 2A.
at pH 9.0 gave almost entirely 13-oxygenation. The F557V mutant gave
a substantial amount of 9-oxygenation at pH 9.0, but the 9-oxygenation
occurred without stereoselectivity and was accompanied by formation
of E,E isomers. These results imply that 9-oxygenation results from
dissociation of the pentadienyl radical, which reacts with O2 in solu-
tion. This interpretation suggests that replacement of phe-557 with
valine either increases the rate at which the pentadienyl radical inter-
mediate dissociates and/or reduces the rate at which the bound inter-
mediate reacts with O2. The second of these possibilities seems less
likely, since the replacement of phenylalanine with a smaller residue is
unlikely to block access of O2 to the bound radical.
Some of our data suggest that replacing phe-557 with valine may
increase the likelihood of dissociation of the pentadienyl radical in the
oxygenation of linoleic acid. The 9-HPOD formed by the mutant from
unlabeled linoleic acid at pH 7.5 has an S/R ratio of 87:13. With 11(S)-
DLA, the S/R ratio of the 9-HOD formed by the mutant is 98:2. The
higher stereoselectivity with 11(S)-DLA suggests that most of the loss of
stereoselectivity that occurs with unlabeled linoleic acid is initiated by
Position-557 in SBLO-1 is reasonably close to a proposed oxygen
channel [21], and the formation of 9(S)-HPOD in the F557V mutant
could possibly result from normal substrate binding and an alteration of
the pathway of oxygen delivery to the pentadienyl radical. However,
this alternative explanation does not account for the fact that 9(S)-
HPOD is formed by loss of the pro-R hydrogen from C-11 of linoleate.
The stereochemical results with 11(S)-DLA are consistent with the
proposal of Hornung et al. [29] that 13(S)-HPOD results from tail-first
binding and 9(S)-HPOD from head-first binding (Scheme 2). However,
the stereochemical results are equally consistent with the possibility
that 13(S)-HPOD results from head-first binding and 9(S)-HPOD from
tail-first binding. The experiments with 10,13-NDA and arachidonic
acid allow a distinction to be made between these two possibilities.
With 10,13-NDA, the extra methylene group between the diene unit
and the carboxy terminus is expected to disfavor the head-first binding
mode and shift the product ratio in favor of the product produced by
tail-first binding. The fact that the relative yield of the 14-ΗPND from
10,13-NDA is higher than that of 13-HPOD from linoleic acid provides
evidence that oxygenation in the ω6 position results from tail-first
binding. This conclusion is also supported by the high ratio of 15-
HPETE to 11-HPETE and 8-HPETE formed by the F557V mutant from
arachidonic acid. If formation of 9-HPOD from linoleic acid involves
head-first binding followed by removal of hydrogen from C-11, for-
mation of 11-HETE from arachidonic acid by head-first binding is ex-
pected to be more difficult, since this process requires removal of hy-
drogen from C-13, which is expected to be poorly positioned for
hydrogen abstraction. The formation of a small amount of 8-HPETE can
be rationalized as arising from head-first binding followed by abstrac-
tion of hydrogen from C-10 and reaction of O2 with C-8 of the penta-
dienyl radical. This process is similar to that depicted in Scheme 2B, in
which head-first binding of linoleic acid is followed by abstraction of
hydrogen from C-11 followed by reaction with the pentadienyl radical
at C-9.
removal of the pro-S hydrogen.
A more complete analysis (see
Supplementary Material) supports this interpretation. In the context of
Scheme 2, this finding implies that dissociation of the pentadienyl ra-
dical is more likely when linoleic acid binds tail-first than when it binds
head-first.
Other groups have found that other mutations of SBLO-1 can change
the regioselectivity of SBLO-1 by mechanisms that are different from
that of F557V. Results from Klinman and coworkers indicate that the
regioselectivity of SBLO-1 can be altered by blocking access of oxygen
to C-13 of the pentadienyl radical [21,58,59], and these results provide
support for the oxygen channel that this group has proposed [21]. Brash
and coworkers [30] found that the A542G mutant converts linoleic acid
to a 56:44 mixture of 13(S)-HPOD and 9(R)-HPOD and that both pro-
ducts result from loss of the pro-S hydrogen. In this case, the stereo-
chemical results imply that the two products result from the same
binding orientation. The authors proposed that the replacement of ala-
542 with the smaller glycine residue allows oxygen greater access to the
re face of C-9 of the pentadienyl radical to give rise to 9(R)-HPOD. Since
similar effects are observed with phospholipid substrates and since ala-
542 is located between the catalytic iron and the surface of the protein,
the results imply that all of the products arise from tail-first binding
[30].
Rickert and Klinman [26] have investigated the action of wild-type
SBLO-1 on 11(S)-DLA and found a large shift in the product ratio in
favor of 9-HPOD. They interpreted this shift as evidence that the 9-
oxygenated product arose from abstraction of the pro-R hydrogen from
C-11 as a result of substrate binding in the opposite orientation as the
one leading to the 13-oxygenated product. Rickert and Klinman de-
picted the 13(S)-HPOD as arising from head-first binding, with the
carboxylate of linoleate interacting with arg-707, and 9-HPOD arising
from the opposite orientation. More recent work from the Klinman
group has modeled the production of 13(S)-HPOD from linoleate as
resulting from tail-first binding [21]; other recent studies model 13(S)-
HPOD as arising from head-first binding [27,38,39]. As noted above,
either interpretation is consistent with the stereochemical results on
11(S)-DLA, as long as 9(S)-HPOD results from binding with the opposite
orientation. Our experiments with 10,13-NDA and arachidonic acid on
the F557V mutant provide evidence that oxygenation in the ω6 position
results from tail-first binding, and additional support is provided by the
regiochemistry of linoleate oxygenation by the F557V,R707L double
mutant. Other evidence for this interpretation includes the ability of
SBLO-1 to catalyze ω6 oxygenation on phospholipids [8,10] and other
large substrates [31,32], and studies by the Brash group on the A542G
mutant [30].
The results reported here demonstrate that the regioselectivity of
SBLO-1 can be altered by a mutation that facilitates an alternative mode
of substrate binding. This work also adds to the body of evidence that
ω6 oxygenation by SBLO-1 results from tail-first binding and ω10
oxygenation from head-first binding.
Declarations of interest
None.
Supplementary Material
Procedures for enzyme purification and expression. Procedures for
the synthesis and enzymatic resolution of racemic 11-deuteriolinoleic
acid. Calculation of percent deuteration from mass spectrometry data.
Stereochemical analysis of products from oxygenation of linoleic acid
and 11(S)-LDA by the F557V mutant. Figs. S1–S7.
Acknowledgments
We thank Dr. Ted Holman, University of California, Santa Cruz, for
providing the plasmid for expression of SBLO-1. Nucleic acid sequen-
cing was carried out at the Penn State Genomics Core Facility,
University Park, PA. This work was funded by grant RUI CHE-1213262
(to C·H.C.) from the National Science Foundation, USA. M.J.P. received
support from grant DUE-1317446 from the National Science
The hypothesis that oxygenation at the ω10 position results from
head-first binding suggests that 9-oxygenation should be very difficult
with phospholipid substrates such as PLPC. In agreement with earlier
work by Brash et al. [8], the oxygenation of PLPC by wild-type SBLO-1
9

D. Hershelman, et al.
ArchivesofBiochemistryandBiophysics674(2019)108082
Foundation, USA.
S. Hammes-Schiffer, J.P. Klinman, B.M. Hoffman, 13C ENDOR spectroscopy of li-
poxygenase-substrate complexes reveals the structural basis for C–H activation by
tunneling, J. Am. Chem. Soc. 139 (2017) 1984–1997.
Appendix A. Supplementary data
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11