Regiochemical and stereochemical evidence for enzyme-initiated catalysis in dual positional specific maize lipoxygenase-1

Article abstract of DOI:10.1021/ol0712024

Dual positional specific maize lipoxygenase-1 catalyzed the formation of racemic mixtures of four possible regioisomers and was strongly inhibited by the radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinoxy radical. Molecular modeling studies indicated that the oxygen-binding cavity is segregated from the substrate-binding cavity. The data suggest that a bis-allylic radical reaction intermediate is generated enzymatically, released from the enzyme active site, and subsequently oxygenated outside of the enzyme active site by a nonenzymatic mechanism.

Full text of DOI:10.1021/ol0712024

ORGANIC
LETTERS
2
007
Vol. 9, No. 16
113-3116
Regiochemical and Stereochemical
Evidence for Enzyme-Initiated Catalysis
in Dual Positional Specific Maize
Lipoxygenase-1
3
Sungkuk Jang, Thavrak Huon, Keumhwa Kim, Eunji Um, and Oksoo Han*
Department of Molecular Biotechnology, Biotechnology Research Institute, College of
Agriculture and Life Sciences, Chonnam National UniVersity, Kwangju 500-757,
Republic of Korea
oshan@chonnam.ac.kr
Received May 22, 2007
ABSTRACT
Dual positional specific maize lipoxygenase-1 catalyzed the formation of racemic mixtures of four possible regioisomers and was strongly
inhibited by the radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinoxy radical. Molecular modeling studies indicated that the oxygen-
binding cavity is segregated from the substrate-binding cavity. The data suggest that a bis-allylic radical reaction intermediate is generated
enzymatically, released from the enzyme active site, and subsequently oxygenated outside of the enzyme active site by a nonenzymatic
mechanism.
2
Lipoxygenases (LOXs) catalyze the hydroperoxidation of
polyunsaturated fatty acids with one or more (1Z,4Z)-
pentadiene structures in the presence of molecular oxygen.
These enzymes play important functions in host defense
formed from LA by 13-LOX. The regioselectivity of 9-LOX
and 13-LOX has been explained by one of two models: the
orientation-determined model (orientation of substrate inser-
tion) or the space-determined model (frame-shift realignment
1
2b
systems in plants. Theoretically, four regioisomers, 13-
of substrate). Evidence indicates that regioselectivity and
stereoselectivity might be linked in LOX reactions, such
3
(9Z,11E)-hydroperoxyoctadecadienoic acid (HPODE) (1),
1
9
3-(9E,11E)-HPODE (2), 9-(10E,12Z)-HPODE (3), and
-(10E,12E)-HPODE (4), can be produced from linoleic acid
that oxygenation occurs on the opposite face (antarafacial)
of the initially abstracted prochiral bis-allylic hydrogen at
one or another end of the bis-allylic radical. However, a few
naturally occurring plant LOXs, including maize LOX1,
exhibit dual positional specificity; these enzymes are clas-
(LA), if the W-conformation of the substrate is maintained
throughout the LOX reaction (Scheme 1). However, most
LOX-catalyzed reactions are highly regioselective and stereo-
selective; for example, in plants, 9(R)-(10E,12Z)-HPODE is
formed from LA by 9-LOX, and 13(S)-(9Z,11E)-HPODE is
(2) (a) Kuhn, H. Prostaglandins Other Lipid Mediators 2000, 62, 255-
70. (b) Feussner, I.; Wasternack, C. Annu. ReV. Plant Biol. 2002, 53, 275-
2
297. (c) L u¨ tteke, T.; Krieg, P.; F u¨ rstenberger, G.; von der Lieth, C. W.
(
1) (a) Brash, A. R. J. Biol. Chem 1999, 274, 23679-23682. (b) Lee, S.
Bioinformatics 2003, 19, 2482-2483.
H.; Ahn, S. J.; Im, Y. J.; Cho, K.; Chung, G. C.; Cho, B. H.; Han, O.
Biochem. Biophys. Res. Commun. 2005, 330, 1194-1198. (c) Nemchenko,
A.; Kunze, S.; Feussner, I.; Kolomiets, M. J. Exp. Bot. 2006, 57, 3767-
(3) (a) Prigge, S. T.; Gaffney, B. J.; Amzel, M. L. Nature: Struct. Biol.
1998, 5, 178-179. (b) Coffa, G.; Imber, A. N.; Maguire, B. C.; Laxmi-
kanthan, G.; Schneider, C.; Gaffney, B. J.; Brash, A. R. J. Biol. Chem.
2005, 280, 38756-38766.
3
779.
1
0.1021/ol0712024 CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/13/2007

specificity was 13-HPODE:9-HPODE ) 53.2:46.8, which
is consistent with our previous report regarding the dual
positional specificity of maize LOX1 with linolenic acid
Scheme 1
4
a
(
LNA) as a substrate.
This is unusual and interesting, because the products of
most LOX reactions, even when carried out by a dual
2c,4b
positional specific LOX, are primarily 1 and 3.
A similar
product distribution is seen during autoxidation of methyl
7
linoleate in benzene. Formation of compounds 1 and 3 is
considered to be kinetically controlled while compounds 2
and 4 are thermodynamically controlled products. Figure 1
shows that maize LOX1 generates compounds 2 and 4 as
well as compounds 1 and 3 from LA. Particularly, the
nonstereoselective nature of this reaction is unique to dual
positional specific maize LOX1, and it implies that oxygen-
ation may occur outside the enzyme active site by a
nonenzymatic mechanism. Therefore, product distribution
was compared for the maize LOX1 reaction and autoxidation
of linoleic acid at different temperatures (Table 1). The
results showed that compounds 1 and 3 were the major
products (78%) at 0 °C (kinetically controlled); in contrast,
all four regiosiomers were formed in more comparable
amounts when the reaction was carried out at 50 °C
sified as nontraditional LOXs, because their patterns of regio-
and stereoselectivity are distinct from traditional LOX
enzymes. Even though issues regarding regioselectivity and
4
stereoselectivity of the LOX reaction have been extensively
5
addressed by many groups, the detailed catalytic mechanism
of the nontraditional dual positional LOX reaction remains
6
controversial. This study analyzes the regio- and stereo-
(thermodynamically controlled). More than 90% of the
selectivity of maize LOX1 and the structure and distribution
of its reaction products. Product analysis and molecular
modeling studies were conducted, and their implications with
regard to an enzyme-initiated catalytic mechanism are
discussed.
products were kinetically controlled compounds 1 and 3
when autoxidation was performed in the frozen state at -20
°
C. Therefore, the product distribution pattern in the maize
LOX1-catalyzed reaction is similar to that of thermodynami-
cally controlled autoxidation. The first and rate-determining
chemical step of the LOX1 reaction involves abstracting the
Recombinant maize LOX1 (free epitope or affinity tags)
was expressed in E. coli and purified by Q-Sepharose
6a,8
prochiral hydrogen from C-11. According to the two-step
4a
chromatography. Purified maize LOX1 was incubated with
LA, the reaction products reduced with TPP and isolated as
9
model, the abstraction of hydrogen from the substrate by
catalytically active Fe(III) produces a bis-allylic radical
intermediate with concurrent reduction of the iron cofactor
to catalytically inactive Fe(II). The second step is insertion
of dioxygen, which is accompanied by reoxidation of Fe(II)
to Fe(III), allowing LOX1 to recycle to an active state. The
two-step model is consistent with the nonstereoselective
outcome of the maize LOX1 reaction, because with such a
mechanism, the allyl radical intermediate could dissociate
from the enzyme active site after the first reaction step. Thus,
oxygenation of the bis-allylic radical intermediate could
subsequently occur outside of the enzyme active site by a
nonenzymatic mechanism.
5
reported previously. Structures of all regioisomers were
identified by GC-MS and NMR. The regioselectivity of
maize LOX1 was estimated from straight phase HPLC (SP-
HPLC), and enantiomeric ratios of each regioisomer were
determined by chiral phase HPLC (CP-HPLC).4 As seen
in Figure 1, maize LOX1 produced all four regioisomers, 1,
a,5
(4) (a) Kim, E. S.; Choi, E.; Kim, Y.; Cho, K.; Lee, A.; Shim, J.; Rakwal,
R.; Agrawal, G. K.; Han, O. Plant Mol. Biol. 2003, 52, 1203-1213. (b)
Hughes, R. K.; West, S. I.; Hornostaj, A. R.; Lawson, D. M.; Fairhurst, S.
A.; Sanchez, R. O.; Hough, P.; Robinson, B. H.; Casey, R. Biochem. J.
2
001, 353, 345-355. (c) Fuller, M. A.; Weichert, H.; Fischer, A. M.;
Feussner, I.; Grimes, H. D. Arch. Biochem. Biophys. 2001, 388, 146-154.
d) Garbe, L.; Almeida, R. B.; Nagel, R.; Wackerbauer, K.; Tressl, R. J.
Agric. Food Chem. 2006, 54, 946-955.
5) Butovich, I. A.; Reddy, C. C. Biochim. Biophys. Acta 2001, 1546,
(
(
3
79-398.
Figure 1. Regiochemical and stereochemical analysis of maize
LOX1 reaction products by SP-HPLC and CP-HPLC.
(6) (a) Knapp, M. J.; Seebeck, F. P.; Klinman, J. P. J. Am. Chem. Soc.
2001, 123, 2931-2932. (b) Cho, K.; Jang, S.; Huon, T.; Park, S.; Han, O.
J. Biochem. Mol. Biol. 2007, 40, 100-106.
(
90.
7) Porter, N. A.; Caldwell, S. E.; Mills, K. A. Lipids 1995, 30, 277-
2
6
(
8) Goldsmith, C. R.; Stack, T. D. P. Inorg. Chem. 2006, 45, 6048-
2, 3, and 4, as racemic mixtures. Product formation was not
055.
significant with the boiled maize LOX1 enzyme under the
employed enzymatic incubation condition. The positional
(9) Schilstra, M. J.; Veldink, G. A.; Vliegenthart, J. F. G. Biochemistry
1994, 33, 3974-3979.
3114
Org. Lett., Vol. 9, No. 16, 2007

Table 1. Comparison of Product Distributions of the Maize LOX1 Reaction with Autoxidation
maize
LOX1 (µg)
temp
(˚C)
1 (S:R)
(%)
3 (S:R)
(%)
KC 1 + 3
2 (S:R)
(%)
4 (S:R)
(%)
TC 2 + 4
(%)
(%)
7
2
0
0
0
25
50
0
29.0 (47.5:52.5)
26.0 (45.6:54.4)
55.0
33.1
78.2
93.4
23.2 (46.2:53.8)
31.4
9.3
21.8 (46.1:53.9)
45.0
66.9
21.8
6.6
16.6
43.5
49.4
16.5
34.7
44.0
35.5
12.5
3.2
-20
3.4
The regiochemical and stereochemical outcome of the
maize LOX1 reaction in Figure 1 and Table 1 strongly
suggest that the bis-allylic radical intermediate could be
exported from the active site to the surface of the enzyme.
If this is in fact the case, it is predicted that the reaction will
be inhibited by a free radical scavenger such as 4-hydroxy-
TEMPO (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinoxy radi-
However, modeling also revealed a striking difference
between the two structures: the oxygen-binding cavity is
disconnected from the substrate-binding cavity in maize
LOX1 as seen in Figure 3, while the oxygen-binding cavity
5
cal). As shown in Figure 2, 4-hydroxy-TEMPO srongly
Figure 3. Substrate channel and oxygen cavity of the maize LOX1
shown as a white surface. The substrate channel is composed of
entrance site and extended cavity.
Figure 2. Inhibition of maize LOX1 by 4-hydroxy TEMPO.
inhibited maize LOX1, such that 0.1 mM 4-hydroxy-TEMPO
reduced the relative enzyme activity by more than 5-fold.
These data strongly support a model in which the bis-allylic
radical reaction intermediate is released from the maize
LOX1 active site prior to the oxygenation step. However,
we cannot exclude the possibility that the maize LOX1 active
site is accessible to 4-hydroxy-TEMPO, which quenches the
bis-allylic radical intermediate before it is released from the
enzyme active site. The latter situation has been reported
is cross-connected to the substrate-binding cavity in soybean
LOX1. Previous studies showed that the oxygen-binding
cavity of soybean LOX1 intersects the substrate-binding
cavity near the point of C-13 for 13-LOX, and the point
where the two cavities intersect determines the regioselec-
6
a
tivity of oxygenation in the reaction. The crystal structure
of the dual positional-specific soybean LOX3 indicated that
the oxygen cavity and the substrate-binding cavity intersect
10
for the manganese-containing LOX.
1
1
over a relatively broad area. This is in marked contrast to
the isolation of the oxygen-binding cavity in maize LOX1,
as reported here. This observation is consistent with a high
probability of dissociation of the bis-allylic radical intermedi-
ate from the enzyme active site of maize LOX1. Therefore,
the separation of the oxygen and substrate binding cavities
in maize LOX1 could provide a structural explanation for
release of the bis-allylic radical intermediate from the enzyme
active site prior to its oxygenation.
Molecular modeling was used to explore the possible
structural basis for the regiochemical and stereochemical
outcome of LOX1. To this end, the three-dimensional
structure of maize LOX1 was modeled by using an auto-
mated mode of the SWISS-MODEL server. Cavities in maize
LOX1 were compared with those of soybean LOX1, a typical
13-LOX, by superimposition of the maize and soybean
LOX1 structures. The modeling studies revealed that nine
residues (Q524, L525, W529, A571, L575, V576, F582,
F593, L783) in the oxygen-binding cavity of maize LOX1
had corresponding residues in soybean LOX1 (i.e., Q495,
L496, W500, A524, L546, L547, I553, V564, and L754).
Maize LOX1 is, to our knowledge, the first example of a
naturally occurring nonstereoselective plant LOX in mono-
cots. Thus, it may be a prototype for a two-step enzyme-
(
11) Youn, B.; Sellhorn, G. E.; Mirchel, R. J.; Gaffney, B. J.; Grimes,
(10) Cristea, M.; Oliw, E. H. J. Biol. Chem. 2006, 281, 17612-17623.
H. D.; Kang, C. Struct. Funct. Bioinformation 2006, 65, 1008-1020.
Org. Lett., Vol. 9, No. 16, 2007
3115

enzyme-initiated first step of the reaction involves hydrogen
abstraction from the substrate, followed by dissociation of
the bis-allylic radical intermediate, which is subsequently
oxygenated at the surface of the enzyme (Scheme 2). This
model is consistent with the dual positional specificity and
nonstereoselectivity of maize LOX1.
Scheme 2
Acknowledgment. This research was supported by
ARPC, the Korea Science and Engineering Foundation (F01-
2
004-000-10124-0), and the Korea Research Foundation
(2005-041-F00016).
Supporting Information Available: Experimental de-
tails, SDS-PAGE of the maize LOX1, structural character-
ization of the products, and modeled structures of maize
LOX1 and soybean LOX1. This material is available free
of charge via the Internet at http://pubs.acs.org.
initiated catalytic mechanism for the oxidation of unsaturated
fatty acids. As stated above, our model proposes that the
OL0712024
3116
Org. Lett., Vol. 9, No. 16, 2007