Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms

Article abstract of DOI:10.1016/j.phytochem.2008.03.002

Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxygenase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phosphatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectivity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not significantly influence the reaction characteristics, while the A162I mutant showed a reduced v_max. Based on the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and a bulkier one in S lipoxygenases at the position that controls stereospecificity.

Full text of DOI:10.1016/j.phytochem.2008.03.002

Phytochemistry 69 (2008) 1832–1837
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Properties of a mini 9R-lipoxygenase from Nostoc sp. PCC 7120 and its mutant forms
Alexandra-Zoi Andreou a, Marian Vanko a,b, Lydia Bezakova b, Ivo Feussner a,
*
a Georg-August-University of Göttingen, Albrecht-von-Haller-Institute of Plant Sciences, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37085 Göttingen, Germany
b
ˇ
Comenius University, Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Kalinciakova 8, 832 32 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipoxygenases (LOXs) consist of a class of enzymes that catalyze the regio- and stereospecific dioxygen-
ation of polyunsaturated fatty acids. Current reports propose that a conserved glycine residue in the
active site of R-lipoxygenases and an alanine residue at the corresponding position in S-lipoxygenases
play a crucial role in determining the stereochemistry of the product. Recently, a bifunctional lipoxyge-
nase with a linoleate diol synthase activity from Nostoc sp. PCC7120 with R stereospecificity and the so far
unique feature of carrying an alanine instead of the conserved glycine in the position of the sequence
determinant for chiral specificity was identified. The recombinant carboxy-terminal domain was purified
after expression in Escherichia coli. The ability of the enzyme to use linoleic acid esterified to a bulky phos-
phatidylcholine molecule as a substrate suggested a tail-fist binding orientation of the substrate. Site
directed mutagenesis of the alanine to glycine did not cause alterations in the stereospecificity of the
products, while mutation of the alanine to valine or isoleucine modified both regio- and enantioselectiv-
ity of the enzyme. Kinetic measurements revealed that substitution of Ala by Gly or Val did not signifi-
cantly influence the reaction characteristics, while the A162I mutant showed a reduced vmax. Based on
the mutagenesis data obtained, we suggest that the existing model for stereocontrol of the lipoxygenase
reaction may be expanded to include enzymes that seem to have in general a smaller amino acid in R and
a bulkier one in S lipoxygenases at the position that controls stereospecificity.
Received 9 December 2007
Received in revised form 6 March 2008
Available online 23 April 2008
Keywords:
Cyanobacteria
Lipid peroxidation
Oxylipin formation
Prokaryotic lipoxygenase
.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and heart disease. Very little is still known about the biological
function of these enzymes in prokaryotes.
Lipoxygenases (LOXs) are a family of structurally related non-
heme iron containing dioxygenases (Brash, 1999). They catalyse
the insertion of molecular oxygen into polyunsaturated fatty acids
(PUFAs) that contain one or more 1,4-pentadiene moieties to give
the corresponding hydroperoxides (Liavonchanka and Feussner,
2006). LOXs occur ubiquitously in plants and mammals, but have
also recently been detected in coral, moss and a number of bacteria
(Kühn et al., 2005; Lang and Feussner, 2007; Liavonchanka and
Feussner, 2006). LOX products can be further metabolized to yield
aldehydes and jasmonates in plants (Feussner and Wasternack,
2002) and lipoxins and leukotrienes in mammals (Samuelsson
et al., 1987; Sigal et al., 1994). These signaling molecules play an
important role in wound healing and defense processes in plants
while in mammals they are involved in inflammation, asthma
LOX classification in plants is done with respect to their posi-
tional specificity of fatty acid oxygenation against linoleic acid
(LA). LA can be oxygenated either at carbon atom 9 (9-LOX) or at
C-13 (13-LOX) of the hydrocarbon backbone, which leads to the
formation of 9-hydroperoxy- and 13-hydroperoxy derivatives of
LA (9- and 13-HPODE), respectively (Liavonchanka and Feussner,
2006). In mammals LOXs are similarly classified according to their
positional specificity of arachidonic acid (AA) oxygenation, which
can take place either at positions C-5 (5-LOX), C-8 (8-LOX), C-9
(9-LOX), C-11 (11-LOX), C-12 (12-LOX) or C-15 (15-LOX) (Schnei-
der et al., 2007). Besides the high regiospecificity, the insertion of
oxygen exhibits also high stereospecificity dependent on the pri-
mary sequence of the enzyme, which is predicted to determine
the orientation and depth of substrate penetration into the active
site (Feussner and Kühn, 2000; Schneider et al., 2007). While the
regiospecificity of LOXs has been the focus of a number of studies,
recent publications describe the importance of a conserved amino
acid in the active site of the enzyme (Coffa et al., 2005). The so
called ‘‘Coffa site” is reported to be a conserved alanine in S-specific
LOXs and a glycine in all R-LOXs. Mutational studies converting the
glycine to an alanine in enzymes of the latter category succeeded
in partially switching the position of oxygenation and chirality of
Abbreviations: ALA, a-linolenic acid; ARA, arachidonic acid; CP-HPLC, chiral
phase-HPLC; HODE, hydroxy octadecadienoic acid; HOTE, hydroxy octadecatrienoic
acid; H(P)ETE, hydro(pero)xy eicosatetraenoic acid; H(P)ODE, hydro(pero)xy octa-
decadienoic acid; LA, linoleic acid; LOX, lipoxygenase; PUFA, polyunsaturated fatty
acid; RP-HPLC, reversed phase-HPLC; SP-HPLC, straight phase-HPLC.
* Corresponding author. Tel.: +49 551 395743; fax: +49 551 395749.
E-mail address: ifeussn@gwdg.de (I. Feussner).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.03.002

A.-Z. Andreou et al. / Phytochemistry 69 (2008) 1832–1837
1833
A
GmLOX-1
AtLOX-1
550
570
638
411
378
170
Hv 13(S)-LOX1
Oc 15(S)-LOX1
NpLOX-1
NspLOXdom
B
Mm 12(R)-LOX
Hs 12(R)-LOX
Ph 8(R)-LOX
449
449
459
Fig. 1. Alignment of DNt-NspLOX with other R and S LOXs. Comparison of NspLOX
(acc. no. NP_478445) from Nostoc sp. with: (A) the S-LOXs GmLOX1 from Glycine
max (acc. no. S25064), AtLOX1 from Arabidopsis thaliana (acc. no. JQ2267), HvLOX1
from Hordeum vulgare (acc. no. U56406), Oc15LOX from Oryctolagus cuniculus (acc.
no. M27214), NpLOX1 from Nostoc punctiforme (acc. no. ZP_00106490) and (B) the
R-LOXs M12(R)-LOX from Mus musculus (acc. no. Y14334), Hs12(R)-LOX from hu-
man (acc. no. AF038461) and the LOX domain of Ph 8(R)-LOX from Plexaura hom-
omalla (acc. no. AF003692). The ‘‘Coffa site”, A in the case of S-LOXs and G in the
case of R-LOXs is highlighted.
the product, thus converting a 13S- into a 9R-LOX enzyme (Coffa
et al., 2005). The only reported exception among S-enzymes is
the mouse platelet-type 12S-LOX which has a serine in this posi-
tion (Coffa and Brash, 2004). Recently, a naturally occurring LOX
with linoleate diol synthase activity from the filamentus cyanobac-
terium Nostoc sp. was reported (Lang et al., 2008). Sequence align-
ment of the protein revealed that an alanine aligned with the
‘‘Coffa site” and therefore suggested that the protein would be a
13S-LOX (Fig. 1). However, oxylipin measurements from Nostoc
sp. PCC7120 as well as expression of the recombinant NspLOX car-
boxy-terminal portion (DNt-NspLOX) exhibited that the protein
harbours a linoleate 9R-LOX activity. The aim of this study is to fur-
ther characterize the enzyme and undertake mutagenesis experi-
ments and examine whether substitution of this alanine with
bulkier and smaller amino acids could alter the stereospecificity.
Fig. 2. SDS–PAGE analysis of the purification DNt-NspLOX. (A) His-trap purifica-
tion: Lane 1, protein ladder; lane 2, BL21[DE3] star cell extracts; lane 3, column
flow-through, lanes 4–7: elution fractions. (B) Lane 1, protein ladder; lane 2, puri-
fied enzyme, after Ni affinity chromatography; lane 3, purified enzyme after gel
filtration. The purified DNt-NspLOX had a molecular weight of 50 kDa as shown by
the single protein band. The experiment is representative for two independent e-
xperiments yielding similar results.
2. Results
2.1. DNt-NspLOX protein purification and kinetic determinations
Table 1
In order to characterize the mini LOX domain of NspLOX (DNt-
NspLOX) in more detail the recombinant DNt-NspLOX was ex-
pressed in BL21 (DE3) star cells and was initially purified using
Ni affinity chromatography (Fig. 2A). Subsequently, the pooled
fractions were further purified with gel filtration to a single 280-
nm UV peak (Fig. 2B). At the end of the purification a unique pro-
tein band could be detected at electrophoretic homogeneity. The fi-
nal protein yield was 10 mg/ml starting from 200 ml of bacterial
expression culture. When the recombinant protein was incubated
either with LA or a-linolenic acid (ALA), which are the fatty acids
that have been previously been detected in Nostoc sp. (Lang
et al., 2008), it converted the substrates to about 90% of (9R)-
HPODE (Table 1 and Fig. 3A) or (9R)-HPOTE, respectively, and
(E,E)-products were only minor products (1–2%). Thus this LOX
produced only kinetically controlled products. The steady state ki-
netic parameters determined (Table 1) indicate that LA binds with
higher affinity at the active site of DNt-NspLOX than ALA, while on
the other hand the catalytic activity (vmax) of the enzyme was al-
most double in the case of ALA in comparison to LA.
Kinetic constants for purified DNt-NspLOX
Parameter
Linoleic acid
a-Linolenic acid
KM
vmax
4.4 lM
31.33 lM/mg protein min
26 lM
53.91 lM/mg protein min
The values given represent the mean values of three independent experiments.
Oxygenation of the fatty acids of dilinoleoyl phosphatidylcholine
(PC) can only be possible when a tail-first binding in the active
site takes place. Purified recombinant protein was incubated
with dilinoleoyl phosphatidylcholine (PC) in the presence of
deoxycholate, the reduced products were transesterified and the
HODE methyl esters were subsequently analysed by HPLC. Four
products could be identified by SP-HPLC analysis: 9-(Z,E)-HODE-
Me (41%) as the main product and additionally 13-(Z,E)-HODE-
Me (29%), 9-(E,E)-HODE-Me (14%) and (D) 13-(E,E)-HODE-Me
(15%) (Fig. 4) and as expected only 9-(Z,E)-HODE-Me was chiral
(data not shown).
2.2. DNt-NspLOX activy with PC as substrate
2.3. Expression and product analysis of wt and mutant DNt-NspLOX
The enzyme’s activity against esterified polyenoic fatty acids
was investigated to establish the substrate binding orientation.
In order to examine whether the amino acid residue aligning
with the ‘‘Coffa site” is also in the case of DNt-NspLOX sufficient

1834
A.-Z. Andreou et al. / Phytochemistry 69 (2008) 1832–1837
mAU
C
2500
2000
1500
1000
500
A
A, WT
R
S
R
S
B
D
0
0
5
5
5
10
10
10
15
20
20
20
25
30
30
30
min
2500
2000
1500
1000
500
min
B, A162V
S
R
Fig. 4. Analysis of products formed by wt DNt-NspLOX incubated with PC. Enzyme
preparations of DNt-NspLOX were incubated with 1,2-dilinoleyl-sn-glycero-3-ph-
osphocholine at pH 7,5. SP-HPLC (here in their reduced form) of 13-(Z,E)-HODE-Me
(peak A), 13-(E,E)-HODE-Me (peak B), 9-(Z,E)-HODE-Me (peak C) and 9-(E,E)-HODE-
Me (peak D).
S
R
Table 2
0
Kinetic constants for purified DNt-NspLOX mutants for LA
0
15
25
Enzyme
KM
vmax
min
Ala ? Gly
Ala ? Val
Ala ? Ile
8.9 lM
5.0 lM
5.6 lM
48.05 lM/mg protein min
35.03 lM/mg protein min
0.27 lM/mg protein min
3000
2500
2000
1500
1000
500
C, A162I
The values given represent the mean values of three independent experiments.
S
R
to Ala exchange is linked to the size difference between the two
amino acids (Coffa and Brash, 2004). We therefore proceeded in
exchanging the Ala residue of DNt-NspLOX into a bulkier amino
acid, such as valine. Indeed the A162V mutant was active and con-
verted LA primarily to 13-HPODE (64%) and to a lesser extent to 9-
HPODE (36%). Chiral analysis by CP-HPLC showed that the main
product was as predicted of predominantly S configuration (94%)
while the secondary product consisted mainly of the R enantiomer
(88%). To investigate whether the substitution of the Ala amino
acid residue by an even bulkier amino acid would further convert
the 9R-LOX to a 13S-enzyme, we replaced Ala by Ile. Analysis of
the reaction products by HPLC showed indeed that this mutant
produced almost exclusively 13-HPODE (90%) in an S configuration
(96%; Table 2).
S
R
0
0
15
25
min
Fig. 3. Analysis of products formed by DNt-NspLOX wild type and ‘‘Coffa site” m-
utant enzymes. Enzyme preparations of wild type (wt), Ala ? Val (A162V) and A-
la ? Ile (A162I) mutants of DNt-NspLOX were incubated with LA at pH 7.5 (here in
their reduced form for SP-analysis). Insets show the analysis of R and S enantiomers
by CP-HPLC analysis. The experiment is representative for two independent expe-
riments yielding similar results.
2.4. Purification and kinetic determination of DNt-NspLOX mutants
to control the stereoselectivity of the reaction we performed site
directed mutagenesis and converted the Ala residue at position
162 to Gly, Val and Ile. Expression of the wild type as well as the
mutant versions of DNt-NspLOX was performed again in BL21 Star
(DE3) cells.
The mutant forms of DNt-NspLOX were purified by His-Trap
affinity chromatography at a final concentration of 1–2 mg/ml to
electrophoretic purity of >95% as judged by SDS-PAGE stained with
Coomassie Brilliant Blue. The purified proteins were used in order
to determine the steady state kinetic parameters of the mutants
and obtain more information on the mechanism that underlies
the observed change of the stereospecificity of the LOX reaction
To investigate the effect of Ala mutation (A162G mutant) on the
stereospecificty of the LOX reaction, cell extracts of Escherichia coli
cells were incubated with LA and the products were analysed by
HPLC (Fig. 3). Wild type DNt-NspLOX produced primarily (9R)-
HPODE (88%) from LA and the stereochemistry of the hydroperox-
ide was almost exclusively R (93%) (Table 2; Fig. 3A). (13S)-HPODE
was only detected in small amounts as by-product. Therefore, the
A162G mutant, the reported determinant amino acid conserved
among R-lipoxygenases, appeared to have no effect on the reaction
product in case of this specific LOX, since the mutant enzyme also
converted LA primarily to (9R)-HPODE. According to the estab-
lished model for regio- and stereospecificity of the LOX reaction,
the change from the 9R- to the 13S-product imposed by the Gly
Table 3
Fatty acid hydroperoxides formed from the reaction of DNt-NspLOX wild type and
mutant enzymes with LA at pH 7.5
Enzyme
9 HODE (R configuration) %
13 HODE (S configuration)
Wild type
Ala ? Gly
Ala ? Val
Ala ? Ile
88 (93)
87 (94)
36 (88)
10 (72)
12 (84)
13 (85)
64 (94)
90 (96)
The values given are mean values from three measurements.

A.-Z. Andreou et al. / Phytochemistry 69 (2008) 1832–1837
1835
upon mutation of the Ala residue aligning with the ‘‘Coffa site”.
Using LA as substrate the mutants had very similar KM to the wt
suggesting that the affinity of the enzyme for the fatty acid remains
unaffected. On the other hand, vmax calculations revealed that
replacement of Ala by Gly or Val did not greatly influence the reac-
tion rate. On the other hand, Ala to Ile mutation resulted in a mu-
tant with a 100-fold smaller rate of LA oxygenation in comparison
with the mutant (Table 3).
ing a 9R-LOX to an enzyme producing 13S-hydroperoxide as major
product. Further substitution of the Ala by the even more space-
filling Ile confirmed this tension and achieved in complete conver-
sion of the enzyme to a 13S-LOX (Table 2). Previous studies have
shown the same tendency upon substitution of the Gly present
in the active site of the 8R-LOX domain of the peroxidase LOX fu-
sion protein from the coral Plexaura homomalla by an Ala (Coffa
and Brash, 2004). Interestingly, in the case of S-LOXs which har-
bour an Ala in the active site, such as human 15-LOX2, replacement
of Ala with a bulkier amino acid such as Val or Thr resulted in an
inactive enzyme (Coffa et al., 2005).
3. Discussion
Kinetic characterization of the mutant enzymes revealed that
substitution of the ‘‘Coffa site” Ala by other amino acids did not
influence affinity of the enzyme for the substrate, as can be seen
by the unaffected KM. The rate of the reaction also appears unaf-
fected in the case of the Ala to Gly and Ala to Val mutation, but
is drastically decreased in the case of the Ala to Ile mutation. Inter-
estingly, this decrease is not apparent in the amount of substrate
converted to the hydroperoxide by crude bacterial extracts
expressing the various proteins (Fig. 3). This observable discrep-
ancy is possibly due to the increased protein amount and reaction
time in the latter experiment.
Whether changes in stereospecificity are strictly connected with
changes in the regiospecificity is still a matter of discussion, since in
the case of the murine 12R-LOX mutation of the ‘‘Coffa site” from a
Gly into an Ala did not significantly change the regiospecificity but
the stereospecificity (Meruvu et al., 2005). In another study muta-
tion of the ‘‘Coffa site” lead to conversion of an 8S- into a 12R-
LOX, respectively, and an 8R- to a 12S-lipoxygenating enzyme (Cof-
fa and Brash, 2004). Our results are in accordance with the latter,
since conversion of the R-LOX DNt-NspLOX to an S producing en-
zyme by mutation of the Ala into an Ile was also accompanied by
a change in the position of oxygenation, namely from C-9 of LA to
The aim of this study was to characterize the minimal LOX do-
main of a bifunctional LOX from Nostoc sp. with respect to its cat-
alytic properties. Therefore the carboxyterminal portion of the
protein was purified to homogeneity after expression in E. coli
and mutagenesis experiments were performed in order to examine
whether substitution of this alanine with bulkier and smaller ami-
no acids could alter the stereospecificity of the enzyme. The steady
state kinetic parameters of the enzyme using the LA and ALA, the
PUFAs found endogenously in Nostoc sp. (Lang et al., 2008), as sub-
strate were determined. The obtained Km for DNt-NspLOX is smal-
ler in comparison to the one measured for plant LOXs from banana
leaf (Kuo et al., 2006), tomato (Todd et al., 1990) and the bacterial
LOX from Thermoactinomyces vulgaris (Iny et al., 1993) and similar
to the one for soybean LOX-1 (Tomchick et al., 2001) and the man-
ganese lipoxygenase from the take-all fungus Gäumannomyces gra-
minis (Su and Oliw, 1998). The difference observed in the vmax of
the reaction when LA and ALA were, respectively, added as sub-
strate also correlates with the increased conversion of the latter
to its product under substrate excess conditions, which has previ-
ously been reported (Lang et al., 2008).
The results presented here also clarify the manner of fatty acid
binding in the active site of DNt-NspLOX. It has been proposed that
in the case of plant lipoxygenases substrate binding is one of the
parameters that determine the regiospecificity of the catalysed
oxygen insertion (Liavonchanka and Feussner, 2006). Enzymes that
insert oxygen at position 9 in the case of LA and 5 in the case of
ARA are thought to bind the fatty acid in ‘‘head first” orientation,
with the carboxyl-end positioned deep in the active site pocket
(Prigge et al., 1996). Taking into account the regiospecificity of
DNt-NspLOX it was especially interesting to establish if the en-
zyme is able to insert molecular oxygen in the fatty acid moieties
of PC, which would only be possible in case of a ‘‘straight” substrate
alignment, as has previously been shown in the case of (15S)-LOX
(Coffa and Brash, 2004). Under high substrate concentration we
were able to detect the multiple reaction products as well. The en-
zyme was able to convert the esterified LA to hydroperoxides, with
9-(Z,E)-HPOD as the main product (Fig. 4), but the specificity of the
reaction is decreased. This suggests that the substrate binding is
suboptimal and that free fatty acids are the preferred substrate.
The stereospecificity of the LOX reaction has been reported to
be controlled by a single amino acid residue (‘‘Coffa site”), which
is a highly conserved Ala in S-LOXs and a the smaller Gly in all
R-LOXs described so far (Coffa and Brash, 2004). However, in a re-
cent study a LOX of cyanobacterial origin which exhibits R-LOX
activity in spite of harbouring an Ala in the ‘‘Coffa site”, was iden-
tified (Lang et al., 2008). In this study, site-directed mutagenesis
was employed in order to examine whether the existing theory
describing stereocontrol of LOX products could be expanded from
a Gly vs. Ala classification into a broader model of a small vs. a
more bulky amino acid depending possibly on the size of the active
site pocket of the enzyme. In agreement with this assumption in
our mutagenesis experiments we found that changing Ala into a
smaller Gly did not influence the stereospecificity of the enzyme,
while substituting Ala with a bulkier valine succeeded in convert-
Fig. 5. A model for the control of stereospecificity in the active site of DNt-NspLOX.
In wt DNt-NspLOX LA may have a tail-first orientation in the active site and the
reaction begins with an abstraction of D-hydrogen. Oxygen attack takes place on the
other side of the molecule. The Ala residue in the ‘‘Coffa site” allows oxygen inse-
rtion at the C-9 of the fatty acid. In the Ala ? Val and Ala ? Ile mutants the subs-
trate binding takes place in the same orientation, but presence of the bulky amino
acids shields the C-9 from oxygen attack and result in C-13 oxygenation.

1836
A.-Z. Andreou et al. / Phytochemistry 69 (2008) 1832–1837
C-13. This suggests that also in the case of DNt-NspLOX the sub-
strate orientation remains unaffected by the performed mutations
and the insertion of a bulkier amino acid (Val and Ile) possibly
shields the C-9 from the oxygen attack (Fig. 5).
150 mM NaCl, 10% (w/w) glycerol, 0.1% (v/v) Tween20). The cells
were disrupted by 2 pulses of 20 s of sonification on ice. 250 lg
of LA was added to a 900 ll aliquot of the crude extract and reac-
tions allowed to proceed for 30 min at room temperature. Hydro-
peroxides formed were reduced to their corresponding
hydroxides with 900 ll of 50 mM SnCl2, dissolved in methanol.
After acidification to pH 3.0 with glacial acetic acid, fatty acids
were extracted as described (Bligh and Dyer, 1959).
However, the low sequence similarity of DNt-NspLOX with
LOXs which have elucidated structures such as LOX-1 and LOX-3
from soybean does not allow modelling of the protein and there-
fore does not allow prediction of the location of the oxygen chan-
nel. Moreover DNt-NspLOX has a large deletion in the protein
sequence, which possibly renders the active site pocket of the pro-
tein different then the majority of LOXs (Lang et al., 2008). It can be
assumed that in the case of DNt-NspLOX the space in the active site
pocket of DNt-NspLOX is larger, since the presence of an Ala in the
position of the ‘‘Coffa site” does not inhibit oxygenation at the C-9.
Additionally the replacement of Ala by significantly more space
filling amino acids such as Val or an Ile can be accommodated
and still yields catalytically active enzymes.
In summary, what can be implied by our results and the com-
parison with previous studies on the stereospecificity of LOXs is
that the residue aligning with a conserved Ala in the case of S-LOXs
and a conserved Gly in R-LOXs is crucial in determining the stereo-
specificity of the product. However, the presence of a Gly vs. an Ala
in the primary structure of a LOX is not sufficient to determine
whether the enzyme is R or S specific, but the morphology and size
of the active site also plays a determining role.
After removing the organic solvent, the residue was reconsti-
tuted in 80 ll of methanol/water/acetic acid (85:15:0.1, v/v) and
subjected to HPLC analysis. HPLC analysis was performed with an
Agilent 1100 HPLC system (Waldbronn) coupled to a diode array
detector. Hydroxy fatty acids were separated from fatty acids by
reversed phase HPLC (RP-HPLC; EC250/4 Nucleosil 120-5 C18,
Macherey-Nagel) eluted with a solvent system of: solvent A, meth-
anol/water/acetic acid (85:15:0.1, v/v) and solvent B, methanol/
acetic acid (100:0.1, v/v) at a flow rate of 0.18 ml/min. Straight
phase HPLC (SP-HPLC) of hydroxy fatty acid isomers was carried
out on a Zorbax SIL column (250/4.6, 5 lm particle size, Agilent)
eluted with a solvent system of hexane/2-propanol/acetic acid
(100:1:0.1, v/v) at a flow rate of 0.2 ml/min. CP-HPLC of the hydro-
xy fatty acids was carried out on a Chiralcel OD-H column
(2.1 ꢀ 150 mm, 5-lm particle size, Daicel, distributed by VWR)
with
a solvent system of n-hexane/2-propanol/acetic acid
(100:5:0.1, v/v) and a flow rate of 0.1 ml/min. The absorbance at
234 nm (conjugated diene system of the hydroxy fatty acids) was
recorded simultaneously during all chromatographic steps.
The activity of DNt-NspLOX using PC as substrate was measured
by incubating 0.7 nmol purified protein with 0.6 mg 1,2 Dilinoleyl-
sn-Glycero-3-Phosphocholine (Avanti Polar Lipids) in 5 ml buffer
(50 mM Tris pH 7.5, 4 mM sodium deoxycholate) under constant
stirring for 30 min at room temperature. Reduction of the hydro-
peroxides was performed as described above. The lipid bound fatty
acids were the converted to the corresponding methyl esters by
adding 500 ll of 1% sodium methoxide solution to the dried after
chloroform evaporation samples and shaking for 20 min at room
temperature. 500 ll 6 M NaCl was added to the reaction and the
methyl esters were then extracted twice with 750 ll hexane. The
solvent was removed by evaporation under streaming nitrogen
and the sample was dissolved in 80 ll of methanol/water/acetic
acid (85:15:0.1, v/v/v). Methyl esters were separated by reversed
phase-HPLC (RP-HPLC) with a solvent system of methanol/water/
acetic acid (75:25:0.1 v/v) at a flow rate of 0.18 ml/min. Straight
phase-HPLC (SP-HPLC) analysis was carried out with a solvent sys-
tem hexane: 2-propanol: trifluoroacetic acid (100:1:0.02, v/v/v) at
a flow rate of 0.1 ml/min.
Note: While this manuscript was under revision another paper
describing a similar set of experiments was published (Zheng et al.,
2008).
4. Experimental
4.1. Site-directed mutagenesis
Construction of mutants of the DNt-NspLOX domain of NspLOX
were performed using the QuikChange site-directed mutagenesis
kit (Stratagene). The overlapping mismatching primers used were
NspLOXA162GF 50-TTATCAACTCATCAGGTGTTCCAAAGATTATC-30;
NspLOXA162GR 50-GATAATCTTTGGAACACCTGATGAGTTGATAA-30,
NspLOXA162VF 50-TTATCAACTCATCAGTTGTTCCAAAGATTATC-30,
NspLOXA162VR 50-GATAATCTTTGGAACAACTGATGAGTTGATAA-30,
NspLOXA162IF
50-TTATCAACTCATCAATTGTTCCAAAGATTATC-30
and NspLOXA162R 50-GATAATCTTTGGAACAATTGATGAGTTGAT-
AA-30. The His-tagged DNt-NspLOX domain in the pET15b expres-
sion vector was used as template (Lang et al., 2008). The desired
mutations were confirmed by sequencing.
4.2. Overexpression and protein purification
4.4. Steady-state kinetic measurements
Expression of the protein was performed in E. coli BL21 Star
(DE3) cells. The cultures were grown at 37 °C until OD600 0.6, in-
duced with 0.1 mM IPTG and further incubated for 48 h at 16 °C.
For purification of DNt-NspLOX frozen cell pellets were lysed using
BPer (Pierce) according to manufacturer’s instructions. Cell debris
was removed by centrifugation at 27,000g for 20 min at 4 °C and
supernatant was applied to a 1 ml HisTrap FF column (GE Health-
care) previously equilibrated with 50 mM Tris pH 7.5. The column
was then washed with 50 mM Tris pH 7.5, 300 mM NaCl, 10 mM
imidazole and protein was eluted with 50 mM Tris pH 7:5,
300 mM NaCl, 200 mM imidazole. DNt-NspLOX was further puri-
fied using the Superdex 200 HR (GE Healthcare).
KM and vmax were determined by monitoring the formation of
the conjugated double bond of the product at 234 nm
(e = 2.5 ꢀ 104 Mꢁ1 cmꢁ1) with CARY 100 Bio (Varian). The assay
samples were 1 ml in volume and substrate concentrations were
3–100 lM (Borate buffer, pH 7.5). Linear parts of the curves were
used for Dixon plot.
Acknowledgements
The authors are grateful to Theres Riemekasten, Göttingen, for
excellent technical assistance and Dr. Cornelia Göbel, Göttingen,
for continuous support and stimulating discussions. A.A. is
supported by the International Max Planck Research School
Molecular Biology (Goettingen) and the project is funded by IRTG
1422 Metal Sites in Biomolecules: Structures, Regulation and
Mechanisms.
4.3. LOX activity assay
For activity assays the pellets of 30 ml expression cultures of E.
coli cells were resuspended in 5 ml lysis buffer (50 mM Tris, pH 8.0,

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1837
Liavonchanka, A., Feussner, I., 2006. Lipoxygenases: occurrence, functions and
catalysis. J. Plant Physiol. 163, 348–357.
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