Calcium modulates membrane association, positional specificity, and product distribution in dual positional specific maize lipoxygenase-1

Article abstract of DOI:10.1016/j.bioorg.2015.04.001

This study investigates how calcium modulates the properties of dual positional specific maize lipoxygenase-1, including its interaction with substrate, association with subcellular membrane and alteration of product distribution. Bioinformatic analyses identified Asp³⁸, Glu¹²⁷ and Glu²⁰¹ as putative calcium binding residues and Leu³⁷ as a flanking hydrophobic residue also potentially involved in calcium-mediated binding of the enzyme to subcellular membranes. Asp³⁸ and Leu³⁷ were shown to be important but not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes in vitro. Kinetic studies demonstrate that catalytic efficiency (V_max/K_m) shows a bell-shaped dependence on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipoxygenase-1 to subcellular membranes.

Full text of DOI:10.1016/j.bioorg.2015.04.001

Bioorganic Chemistry 60 (2015) 13–18
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Calcium modulates membrane association, positional specificity, and
product distribution in dual positional specific maize lipoxygenase-1
a
a
b
a,
⇑
Kyoungwon Cho , Jihoon Han , Randeep Rakwal , Oksoo Han
a
Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757,
Republic of Korea
Organization for Educational Initiatives, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8577, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 December 2014
Available online 9 April 2015
This study investigates how calcium modulates the properties of dual positional specific maize lipoxy-
genase-1, including its interaction with substrate, association with subcellular membrane and alteration
38
127
201
of product distribution. Bioinformatic analyses identified Asp , Glu
and Glu
as putative calcium
binding residues and Leu3 as a flanking hydrophobic residue also potentially involved in calcium-medi-
7
Keywords:
Lipoxygenase
Calcium
Linoleic acid
Dual positional specificity
Monocot
38
37
ated binding of the enzyme to subcellular membranes. Asp and Leu were shown to be important but
not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes
m
in vitro. Kinetic studies demonstrate that catalytic efficiency (Vmax/K ) shows a bell-shaped dependence
on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of
the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount
of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction
by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipox-
ygenase-1 to subcellular membranes.
Ó 2015 Elsevier Inc. All rights reserved.
1
. Introduction
characterized [7]. Although the catalytic mechanism of dual posi-
tional specific LOX enzymes remains controversial [8], we provided
Lipoxygenases (LOXs) (EC 1.13.14.12) catalyze the hydroperox-
idation of polyunsaturated fatty acids (PUFA) with one or more (1Z,
Z)-pentadienes. Most LOX-catalyzed reactions are regioselective
and stereoselective. For example, linoleic acid (LA) and linolenic
acid (LNA) are oxygenated by 9-LOX at C-9 or 13-LOX at C-13 of
regiochemical and stereochemical evidence that ZmLOX1 acts by a
unique enzyme-initiated catalytic mechanism [5,6].
4
The specific functions of calcium in LOX have been thoroughly
investigated in animals and calcium was shown to bind to LOX,
mediate its association with membranes, and activate the LOX
activity [9–11]. The crystal structure of a calcium-dependent LOX
revealed that it is composed of two domains: a C-terminal domain
containing the catalytic site and an N-terminal C2-like domain
the
hydroperoxyoctadecadienoic (trienoic) acid (HPOD(T)E) or (13S)-
9Z,11E)HPOD(T)E, respectively [1,2]. However, the positional
hydrocarbon
backbone,
generating
(9S)-(10E,12Z)
(
specificity of some LOX enzymes is dual specific and does not agree
with the prediction based on specificity motifs [3,4]. Four of those
LOX enzymes identified to date are expressed in monocot plants
and might not be regarded as classical LOX [3–5]. One of these,
maize lipoxygenase-1 (ZmLOX1) has dual positional specificity
known as PLAT (Polycystin-1, Lipoxygenases and Alpha Toxin)
domain. Molecular basis for the interaction of calcium regulating
membrane binding and stimulation of activity has been proposed
[
9,11]. In plants, calcium is known to play roles in senescence
and defense against pathogens, and a possible role for calcium in
oxidation of PUFA has been proposed [12]. Although the activity
of plant LOX enzymes was initially thought to be calcium-indepen-
dent, it has been reported that calcium regulates association of
soybean LOX to subcellular membranes [13] through an interaction
with the N-terminal domain of LOX, which also regulates catalytic
activity [14,15]. ZmLOX1 is involved in pathogen defense and in
activating responses to oxidative stress [16]. Here, we propose that
calcium may play important roles in regulating the activity of
[
6] and its pre-steady state kinetic behavior has been extensively
Abbreviations: HOT(D)E, hydroxyoctadecatri(di)enoic acid; HPOT(D)E, hydro-
peroxyoctadecatri(di)enoic acid; KC, kinetically-controlled; KODE, keto-octadeca-
dienoic acid; LA, linoleic acid; LNA, linolenic acid; LOX, lipoxygenase; PLAT,
Polycystin-1, Lipoxygenases and Alpha Toxin; PUFA, polyunsaturated fatty acid; TC,
thermodynamically controlled; ZmLOX1, maize lipoxygenase-1..
⇑
Corresponding author. Fax: +82 62 530 2049.
E-mail address: oshan@chonnam.ac.kr (O. Han).
http://dx.doi.org/10.1016/j.bioorg.2015.04.001
045-2068/Ó 2015 Elsevier Inc. All rights reserved.
0

1
4
K. Cho et al. / Bioorganic Chemistry 60 (2015) 13–18
ZmLOX1. In fact, we previously reported that ZmLOX1, when over-
expressed in the cytoplasm of transgenic rice cells, associated with
the chloroplast membrane in a calcium-dependent manner [17]. In
this paper, evidence is provided that calcium facilitates the sub-
cellular membrane association of ZmLOX1, promotes binding to
LA, and alters the product distribution of reaction products during
oxidation of LA.
centrifugation at 12,000 g and the pellet resuspended in an equal
volume of the same buffer. An aliquot (20 L) of the supernatant
and solubilized pellet was analyzed by 10% SDS–PAGE followed
l
by immunoblot as described previously [17].
2.4. Kinetic studies of ZmLOX1 in the presence and absence of calcium
Spectral analysis of the oxidation of LA by ZmLOX1 in the pres-
2
. Materials and methods
ence of calcium was performed as follows. ZmLOX1 reaction was
initiated by adding purified ZmLOX1 (6 g) to reaction buffer, con-
taining 50 mM Tris–HCl (2.5 mL), pH7.2, 0.5 mM LA, 0.05% Tween
20, 100 M EGTA without or with 200 M CaCl . UV spectra
210–350 nm) were recorded every 2 min for 20 min. The concen-
trations of HPODE and KODE were calculated using the extinction
l
2.1. Bioinformatic analysis: modeling, structural analysis, and
identification of calcium binding site of ZmLOX1
l
l
2
(
Three-dimensional structure of ZmLOX1 was modeled using the
SWISS-MODEL ExPASy server (http://swissmodel.expasy.org) in
automated mode. From known X-ray coordinates of LOXs [5], soy-
bean LOX1 (Protein Data Bank accession code: 1yge, 1.4 Å res-
olution) was identified as a suitable template for the structure of
ZmLOX1 (Gene bank accession code: AAF76207). Energy minimiza-
tion was carried out with GROMOS. The quality of the ZmLOX1
structural model was evaluated using PROCHEK (SWISS-MODEL
ExPASy server). Sequences of soybean LOX1, soybean LOX3, and
ZmLOX1 were aligned and the secondary structures were analyzed
using iMolTalk v3.1 (http://i.moltalk.org). PyMOL v0.99 (http://py-
mol.sourceforge.net) was used to superimpose ZmLOX1 and soy-
bean LOX1 models, and calcium binding residues of soybean
LOX1 were identified according to the previously reported method
À1
À1
À1
À1
coefficients of 25,000 M cm at 234 nm and 22,000 M cm at
280 nm, respectively.
Kinetic studies of ZmLOX1 in the presence and absence of cal-
cium were performed as follows. The enzymatic activity of purified
ZmLOX1 was quantified by monitoring absorbance at 234 nm in
variable concentrations of LA (0.05, 0.1, 0.25, 0.5, 0.75 and 1 mM)
and calcium chloride (0, 1, 10, 25, 50, 75, 100, 120, 150, 200, 400,
and 600
modifications. The reaction solution contained 50 mM Tris–HCl
(2.5 mL), pH 7.2, 100 M EGTA, and various concentrations of LA/
lM). Assays were conducted as described [7] with minor
l
Tween 20 (with molar ratio of 3.07) and calcium chloride. The reac-
tion mixture was pre-incubated for 5 min at 25 °C and then the
reaction was initiated by adding 6
lg purified ZmLOX1. The con-
[
13]. Putative calcium-binding residues in ZmLOX1 were predicted
centration of free calcium in the reaction mixture was calculated
2
+
based on structural homology and alignment of the two proteins
[
compose calcium binding site I were used to predict corresponding
amino acid residues in ZmLOX1.
using a Ca /EGTA calculator (http://entropy.brneurosci.org/egta.
html). Curves were fitted using the Boltzmann equation in
OriginPro 7.5 program and initial rates were calculated from the
linear portion of the curves. ZmLOX1 activity was plotted against
concentration of free calcium at various concentrations of LA; plots
2
1
106
179
17,18] and Glu , Glu
and Glu
residues of soybean LOX1 that
2
+
of log[LA]/[Ca
]free were analyzed using LAB Fit Curve Fitting soft-
2
.2. Overexpression and purification of ZmLOX1 and N-terminal
ware v7.2.37 (http://zeus.df.ufcg.edu.br/labfit/), which determined
truncated ZmLOX1
2
the best fit equation to be [y = (a + x)/(b + cx )]. Kinetic parameters
ZmLOX1 and N-terminal truncated ZmLOX1 (lacking Met1 to
(K , Vmax) were obtained by Hanes-Woolf plots, where the ratio of
LA to free calcium was 0.3 to 10,000, at various concentrations of
LA (0.05–1 mM).
m
Cys60) were expressed in E. coli, purified as described previously
[
17]. Briefly, recombinant pRSETB-ZmLOX1 and pRSETB-N-term-
inal truncated ZmLOX1 plasmids were transformed into
BL21(DE3)pLysS, induced using IPTG, and cell extracts were pre-
pared for enzyme purification by Q-Sepharose chromatography.
Purified enzymes were analyzed by SDS–PAGE.
3
. Results
3.1. Putative calcium binding residues in ZmLOX1 and calcium-
dependence of membrane association
2.3. Preparation of subcellular membrane fraction and analysis of
membrane association
Sequence alignment, homology search and structural modeling
were used to identify two protein domains in ZmLOX1, both of
which are typical domains in the LOX family (Fig. 1). The N-term-
inal domain (residues 23–170) is a conserved eight-stranded b-
barrel known as a PLAT domain. The C-terminal catalytic domain
A subcellular membrane fraction was isolated from leaves of
one month-old rice plants (Oryza sativa L. Japonica cv. Nakdong)
by the previously reported method as follows [19]. Rice leaves
(
5 g) were homogenized at 10,000 g for 2 min in 10 mM KH
2
PO
4
(residues 171–873) of ZmLOX1 contains 21 a-helixes, as does the
121
buffer (20 mL), pH 7.8, with 0.5 M sucrose and 1 mM EDTA. The
homogenate was filtered through four layers of gauze and cen-
trifuged at 1,000 g for 30 min. The supernatant was centrifuged
again at 12,000 g for 1 h. The pellet was washed 6 times with
C-terminal domain of soybean LOX1 and LOX3. Lysine (Lys ) in
b5 of the ZmLOX1 structural model corresponds to Lys1 in b5
of soybean LOX1, a residue thought to be involved in membrane
association [18,21]. Superimposition of ZmLOX1 and soybean
00
3
8
127
201
1
mM EDTA to remove any metal species and washed again with
LOX1 suggests that Asp , Glu
and Glu
residues of ZmLOX1
2
1
106
179
PBS buffer several times and stored at 4 °C. The affinity of
ZmLOX1 for the subcellular membrane fraction was determined
as follows [20]. Subcellular membranes (1 mg/mL, Fresh weight)
were pelleted by centrifugation at 12,000 g for 30 min and resus-
are structurally equivalent to Glu , Glu
and Glu
residues of
soybean LOX1 that play a role in calcium binding in soybean
LOX1 (Fig. 2, Fig. S1). Calcium-binding residues in soybean LOX1,
LOX3, VLX-B and VLX-D, are invariably flanked to the N-terminal
side by a hydrophobic residue (i.e., leucine), of which side chain
intercalates into the membrane [13]. The equivalent residue in
pended in 0.5 mL of 10 mM KH
mM, 10 M, 100 nM, or 1 nM calcium chloride. The membrane
suspension (180 L) was mixed with 13.8 g of ZmLOX1 (or N-
2 4
PO buffer, pH 7.8, containing
1
l
3
7
l
l
ZmLOX1 is Leu .
terminal truncated ZmLOX1) and incubated for 5 min at room tem-
perature. The supernatant was separated from the pellet by
To examine the functional roles of the proposed residues in
ZmLOX1, an expression construct was prepared to overexpress

K. Cho et al. / Bioorganic Chemistry 60 (2015) 13–18
15
Fig. 1. Sequence alignment and secondary structure of ZmLOX1. The alignment of sequences of ZmLOX1 (GenBank accession no. Af271894), soybean LOX1 (PDB code no:
YGE, 1.4 Å resolution) and soybean LOX3 (1RRL, 2.09 Å) were automatically performed in SWISS Model Server (http://swissmodel.expasy.org/). The secondary structure of
1
modeled ZmLOX1 was analyzed by iMolTalk v3.1 (http://i.moltalk.org/).
Fig. 2. Calcium-binding sites of ZmLOX1 and soybean LOX1 revealed by three-dimensional superimposition. Three dimensional structure of ZmLOX1 was modeled by an
automated mode of SWISS-MODEL sever (http://swissmodel.expasy.org) using known X-ray coordinates of soybean LOX1 (Protein Data Bank accession code: 1yge, 1.4 Å
resolution) as the best template, secondary structures of soybean LOX1 and ZmLOX1 were analyzed by iMolTalk v3.1 (http://i.moltalk.org), and superimposed to reveal
21
106
179
residues within the calcium binding site as described in Materials and methods. (A) The calcium-binding residues (Glu , Glu1 , Glu ) in soybean LOX1. (B) The calcium-
binding residues (Asp38, Glu
127
and Glu ) in ZmLOX1.
201
N-terminally truncated ZmLOX1 (amino acid 61 to 873), which
fraction was weaker than the affinity of full-length ZmLOX1, espe-
cially at low concentrations of calcium (Fig. 3). Similar result was
obtained with His-tagged LOX (Fig. S2). This result demonstrates
that calcium promotes association of ZmLOX1 to subcellular mem-
branes via its N-terminal PLAT domain in vitro. This result is consis-
tent with our previous observation that calcium mediates
translocation of recombinant ZmLOX1 from cytoplasm to chloro-
plast, when it is overexpressed in transgenic rice [17].
3
8
37
lacks Asp and Leu , but retained a normal level of catalytic activ-
ity as reported previously [17]. The affinity of the purified proteins
for a subcellular membrane fraction was then evaluated in vitro in
the presence of various concentrations of calcium. The results
show that calcium promotes dose-dependent association of
ZmLOX1 to a purified subcellular membrane fraction in vitro, but
the affinity of N-terminal truncated ZmLOX1 for the membrane

1
6
K. Cho et al. / Bioorganic Chemistry 60 (2015) 13–18
presented above (Fig. 6) suggests that calcium interacts with both
ZmLOX1 and LA, which implied that calcium might modulate the
positional specificity of ZmLOX1. Table 1 demonstrate that calcium
increased relative proportions of 13-isomers and kinetically con-
trolled (KC) products. Therefore, calcium modulates the positional
specificity and product distribution during ZmLOX1-catalyzed oxi-
dation of LA.
4
. Discussion
Calcium can stimulate LOX activity and promotes interaction
with membrane lipids by forming salt bridges between acidic pro-
tein residues of LOX and negatively-charged lipids in the mem-
brane [13,24]. The PLAT domain is conserved in LOXs and known
to promote protein-lipid interactions in a calcium-dependent man-
ner [21]. The PLAT domain of ZmLOX1 encompasses putative
Fig. 3. Dependence of membrane association on calcium of ZmLOX1 and N-
terminal truncated LOX1. Subcellular membranes were prepared from rice leaves
and mixed with enzymes, and the degree of membrane association was analyzed by
immunoblot as described in Materials and methods. (A) N-terminal truncated
ZmLOX1 and (B) ZmLOX1.
3
8
127
amino acid residues for binding to calcium (Asp , Glu
and
2
01
121
Glu ) and membrane lipids (Lys ) (Fig. 2). However, results in
Fig. 3 indicate that the N-terminal truncated ZmLOX1 lacking
3.2. Effect of calcium on kinetics of oxidation of LA by ZmLOX1
1
60
Met to Cys still interacts with subcellular membrane fractions
in vitro, albeit with reduced affinity (Fig. 3 and Fig. S2). We con-
The oxidation of LA by ZmLOX1 was examined in the presence
37
38
clude that Leu and Asp play important but non-essential roles
in calcium-mediated binding of ZmLOX1 to subcellular mem-
branes. This in turn suggests that calcium-binding residues
and absence of calcium, and reaction kinetics were analyzed.
Results indicate that calcium stimulates production of HPODE
and KODE (keto-octadecadienoic acid) from LA (Fig. 4A and
Fig. 4B) [22,23], and that the induction period of ZmLOX1 [7] is
altered by calcium (Fig. S3). Although stimulation of ZmLOX1
activity by calcium increases with increasing concentration of LA
127
201
121
Glu
and Glu
and Lys
in the PLAT domain may play a domi-
nant role in promoting association between ZmLOX1 and sub-
cellular membranes.
The ability of calcium to stimulate LOX activity has been
reported previously [25–28]. Evidence provided here and else-
where demonstrates that calcium may regulate ZmLOX1-catalyzed
oxidation of LA by more than one mechanism. Calcium interacts
with fatty acid substrates of LOX, modulating their physical state
in solution [29], and increasing accessibility of substrate to
enzyme. In addition, calcium can activate enzymes by forming an
Enzyme-Metal-Substrate or Enzyme-Substrate-Metal bridge [30].
(
Fig. 5A), the reaction reached a plateau and then declined slightly,
indicating inhibition by high concentration of free calcium. This
suggests that the effect of calcium on the oxidation of LA by
ZmLOX1 depends on the concentrations of both calcium and LA.
To explore this further, ZmLOX1 activity was analyzed as a func-
2
+
tion of log [LA]/[Ca
activity is maximal when log [LA]/[Ca
depending on concentration of LA). A similar analysis of Vmax
]free (Fig. 5B). This plot reveals that ZmLOX1
2+
]
free is in the range 1 to 3
(
Our data indicate that calcium-mediated stimulation of oxidation
+
and K
max of ZmLOX1 decreases as [LA]/[Ca2+]free increases with an
inverted bell-shape curve of K , while a plot of catalytic efficiency
free results in a bell-shaped curve
m
as a function of log [LA]/[Ca2
]free (Fig. 6A) shows that
2+
of LA by ZmLOX1 is strongly dependent on log [LA]/[Ca
]
free
V
(Fig. 5). Calcium reduced the induction period of the ZmLOX1 reac-
m
tion (Fig. S3, which could reflect oxidation of Fe(II) to Fe(III) in the
active site of the enzyme by hydroperoxy fatty acid [31]. In addi-
m
Vmax/K ]
) vs log [LA]/[Ca2
+
(
(
Fig. 6B).
tion, Vmax and K
m
of ZmLOX1 strongly depend on log [LA]/
free (Fig. 6A) and a plot of catalytic efficiency (Vmax/K ) vs
free generates a bell-shaped curve (Fig. 6B), while
2
+
[
Ca
log [LA]/[Ca
vs log [LA]/[Ca ]free generates an inverted bell-shaped curve
]
m
2
+
3
.3. Effect of calcium on positional specificity and product distribution
]
2
+
K
m
The dual positional specificity and lack of stereochemical speci-
ficity of ZmLOX1-catalyzed oxidation of LA has been explained by
invoking an enzyme-initiated catalytic mechanism [5,6]. Data
(Fig. 6A). Therefore, we propose that calcium could bind the
HPODE reaction product and promote reoxidation of Fe(II), which
could in turn enhance the enzyme efficiency.
Fig. 4. Effect of calcium on the oxidation of LA by ZmLOX1. ZmLOX1 reaction was initiated by adding 6
Tween 20, 100 M EGTA. Spectra of ZmLOX1 reaction mixtures were recorded every 2 min for 20 min in the presence of 200
using the extinction coefficients 25,000 M cm at 234 nm and 22,000 M cm at 280 nm, respectively. (A) Quantification of HPODE and (B) quantification of KODE.
lg purified ZmLOX1 in 50 mM Tris–HCl (pH 7.2), 0.5 mM LA, 0.05%
l
l
M CaCl . HPODE and KODE were quantified
2
À1
À1
À1
À1

K. Cho et al. / Bioorganic Chemistry 60 (2015) 13–18
17
Fig. 5. Dependence of ZmLOX activity on concentrations of LA and calcium. (A) Dependence of ZmLOX1 activity on free calcium. Absorbance of product was monitored at
34 nm in the presence of the indicated concentration of LA (0.05, 0.1, 0.25, 0.5, 0.75 and 1 mM) and CaCl (0, 1, 10, 25, 50, 75, 100, 120, 150, 200, 400, 600 M). The activity of
2
2
l
2+
ZmLOX1 was measured and the concentration of free free calcium was calculated using on-line Ca /EGTA calculator (http://entropy.brneurosci.org/egta.htmL) as described
in Materials and methods. (B) Dependence of ZmLOX1 activity on log [LA]/[Ca2
Fitting software v7.2.37 (http://zeus.df.ufcg.edu.br/labfit/).
+
]
free. The data points were analyzed using the equation [y = (a + x)/(b + cx )] and LAB Fit Curve
2
Fig. 6. Dependence of ZmLOX1 kinetic parameters on log [LA]/[Ca2+
]free. Kinetic parameters (K , Vmax) were obtained by Hanes-Woolf plot using ratios of 0.3 to 10,000 LA to
m
on log [LA]/[Ca2
+
]free. (B) Dependence Vmax/K on log [LA]/[Ca ] .
2+
free calcium at the indicated concentrations of LA (0.05–1 mM). (A) Dependence of Vmax and K
m
m
f
r
e
e
Table 1
a
Effect of calcium on product distribution and positional specificity.
[
Ca2+
]free (mM) Product distribution
Positional specificity
KCb vs TCc
13-(9E,11Z)HOD (1) 13-(9E,11E)HOD (2) 9-(10E,12Z)HOD (3) 9-(10E,12E)HOD (4) 13-HOD (1 + 2) 9-HOD (3 + 4) KC (1 + 3) TC (2 + 4)
0
1
3
35.5 ± 0.3
43.8 ± 0.4
39.0 ± 0.3
13.0 ± 0.1
8.8 ± 0.1
16.0 ± 0.1
34.4 ± 0.3
38.3 ± 0.3
36.0 ± 0.3
17.2 ± 0.1
9.1 ± 0.1
9.0 ± 0.1
48.5 ± 0.4
52.6 ± 0.5
55.0 ± 0.4
51.6 ± 0.4
47.4 ± 0.4
45.0 ± 0.4
69.9 ± 0.6 30.2 ± 0.2
82.1 ± 0.7 17.9 ± 0.2
75.0 ± 0.6 25.0 ± 0.2
.5
1.0
a
b
c
The value is the percentage of each regioisomeric form to total HOD products (average and standard error of two replications).
KC: kinetically controlled products.
TC: thermodynamically controlled products.
The positional specificity of other dual positional specific LOX
(Table 1). In addition, the accessibility of substrate to ZmLOX1 is
sensitive to the ratio of LA to free calcium, which results in the
bell-shaped dependence of catalytic efficiency on log [LA]/
enzymes (pea 9/13 LOX and potato 13/9-LOX) depends on the
degree of penetration of the methyl terminus of LA into the
enzyme active site [32,33]. In the case of ZmLOX1, we previously
reported that ZmLOX1 generates four regioisomeric products of
LA, which has been explained by postulating an enzyme-initiated
catalytic mechanism [6]. Here, we provide evidence that calcium
modulates the positional specificity and distribution of products
of the ZmLOX1 reaction (Table 1). Most classical 9-LOX and 13-
LOX enzymes produce only KC products because the (1Z, 4Z)-con-
figuration of substrate is retained in the active site during the cat-
alytic cycle. The enzyme-initiated catalytic mechanism of ZmLOX1
predicts that calcium would influence the distribution of between
KC and thermodynamically-controlled (TC) products. In particular,
the presence of calcium favors kinetically-controlled (E,Z)-isomeric
products over thermodynamically-controlled (E,E)-isomers
2
+
[Ca
]free (Fig. 6B). Accessibility of substrate to the enzyme active
site might be enhanced through an electrostatic interaction
between cationic calcium ion and anionic fatty acid substrate of
LOX1, although the order of interaction between enzyme, calcium
and substrate remains unknown.
In summary, the results presented here suggest that calcium
binds to and promotes binding of LA to ZmLOX1, activates the
enzyme, and may participate in an Enzyme-Metal-Substrate
bridge. The bell-shaped (or inverted bell-shaped) relationship
between kinetic parameters of ZmLOX1 and log [LA]/[Ca2+
]free are
likely to reflect the combined influence of calcium on the above-
mentioned properties of ZmLOX1, and the possibility that calcium
may also interact with ZmLOX1 reaction products.

1
8
K. Cho et al. / Bioorganic Chemistry 60 (2015) 13–18
[
[
[
12] S. Rosahl, Z. Naturforsch C. 51 (1996) 123–138.
13] S.A. Tatulian, J. Steczko, W. Minor, Biochemistry 37 (1998) 15481–15490.
14] J.M. Lopez-Nicolas, R. Bru, J. Agric. Food Chem. 48 (2000) 292–296.
Acknowledgment
This work was supported by a National Research Foundation
grant (2011-0008591) to O. Han.
[15] M. Maccarrone, M.L. Salucci, G. Zadelhoff, F. Malatesta, G. Veldink, J.F.G.
Vliegenthart, A. Finazzi-Agro, Biochemistry 40 (2001) 6819–6827.
[
16] K. Cho, Y.C. Kim, J.C. Woo, R. Rakwal, G.K. Agrawal, S. Yoeun, O. Han, Plant Sci.
86 (2012) 238–245.
1
Appendix A. Supplementary material
[17] K. Cho, Y. Han, J.C. Woo, B. Baudisch, R.B. Klösgen, S. Oh, J. Han, O. Han, Plant
Sci. 181 (2011) 242–248.
[
18] B. Youn, G.E. Sellhorn, R.J. Mirchel, B.J. Gaffney, H.D. Grimes, C. Kang, Proteins
5 (2006) 1008–1020.
[19] S. Munné-Bosch, L. Alegre, Plant Physiol. 125 (2001) 1094–1102.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.04.
6
[
20] R. Brinckmann, K. Schnurr, D. Heydeck, T. Rosenbach, G. Kolde, H. Kühn, Blood
1 (1998) 64–74.
21] A. Bateman, R. Sandford, Curr. Biol. 9 (1999) R588–R590.
0
01.
9
[
References
[22] H. Kuhn, R. Wiesner, J. Rathmann, T. Schewe, Eicosanoids 4 (1991) 9–14.
23] I. Ivanov, J. Saam, H. Kuhn, H.G. Holzhütter, FEBS J. 272 (2005) 2523–2535.
[
[
[
1] I. Feussner, H. Kuhn, C. Wasternack, Plant Sci. 6 (2001) 268–273.
2] T. Lütteke, P. Krieg, G. Fürstenberger, C.W. Von der Lieth, Bioinformatics 19
[24] A. Baba, S. Sakuma, H. Okamoto, T. Inoue, H. Iwata, J. Biol. Chem. 264 (1989)
15790–15795.
[25] R.T. Holman, P.O. Egwin, W.W. Christie, J. Biol. Chem. 244 (1969) 1149–1511.
[26] J. Christopher, E. Pistorius, B. Axehod, Biochim. Biophys. Acta 284 (1972) 54–
62.
[27] W.M. Verhue, A. Francke, Biochem. Biophys. Acta 284 (1972) 43–53.
[28] T.D. Dressen, U. Dickens, R.B. Koch, Lipids 17 (1982) 964–969.
[29] D.C. Zimmerman, H.E. Snyder, J. Agric. Food Chem. 22 (1974) 802–805.
[30] A.S. Mildvan, Metals in enzyme catalysis, in: P.D. Boyer (Ed.), The Enzymes,
New York Academic Press, 1970, pp. 445–536.
[31] M.J. Schilstra, G.A. Veldink, J.F.G. Vliegenthart, Biochemistry 32 (1993) 7686–
7691.
[32] R.K. Hughes, D.M. Lawson, A.R. Hornostaj, S.A. Fairhurst, R. Casey, Eur. J.
Biochem. 268 (2001) 1030–1040.
[33] R.K. Hughes, S.I. West, A.R. Hornostaj, D.M. Lawson, S.A. Fairhurst, R.O.
Sanchez, P. Hough, B.H. Robinson, R. Casey, Biochem. J. 353 (2001) 345–355.
(
2003) 2482–2483.
[
[
3] I. Feussner, C. Wasternack, Annu. Rev. Plant Biol. 53 (2002) 275–297.
4] E. Kim, E. Choi, Y. Kim, K. Cho, A. Lee, J. Shim, R. Rakwal, G.K. Agrawal, O. Han,
Plant Mol. Biol. 52 (2003) 1203–1213.
5] T. Huon, S. Jang, K. Cho, R. Rakwal, J.C. Woo, I. Kim, S.W. Chi, O. Han, Bull.
Korean Chem. Soc. 30 (2009) 917–923.
[
[
[
6] S. Jang, T. Huon, K. Kim, E. Um, O. Han, Org. Lett. 9 (2007) 3113–3116.
7] K. Cho, S. Jang, T. Houn, S. Park, O. Han, J. Biochem. Mol. Biol. 40 (2007) 100–
1
06.
[
[
8] M.J. Knapp, F.P. Seebeck, J.P. Klinman, J. Am. Chem. Soc. 123 (2001) 2931–2932.
9] T. Hammarberg, P. Provost, B. Persson, O. Radmark, J. Biol. Chem. 275 (2000)
3
8787–38793.
[
[
10] M. Walther, R. Wiesner, H. Kuhn, J. Biol. Chem. 279 (2004) 3717–3725.
11] P. Eek, R. Järving, I. Järving, N.C. Gilbert, M.E. Newcomer, N. Samel, J. Biol.
Chem. 287 (2012) 22377–22386.