Structural Basis for Lipoxygenase Specificity
CONVERSION OF THE HUMAN LEUKOCYTE 5-LIPOXYGENASE TO A
15-LIPOXYGENATING ENZYME SPECIES BY SITE-DIRECTED
MUTAGENESIS*
Kristin
Schwarz
,
Matthias
Walther
,
Monika
Anton
,
Christa
Gerth
,
Ivo
Feussner§, and
Hartmut
Kuhn
¶
From the
Institute of Biochemistry, University
Clinics Charité, Humboldt-University, Hessische Strasse 3-4,
D-10115 Berlin and the § Institute of Plant Biochemistry,
Weinbergweg 3, D-06120 Halle, Germany
Received for publication, June 13, 2000, and in revised form, August 31, 2000
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ABSTRACT |
Mammalian lipoxygenases constitute a
heterogeneous family of lipid-peroxidizing enzymes, and the various
isoforms are categorized with respect to their positional specificity
of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases.
Structural modeling suggested that the substrate binding pocket of the
human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket
was reduced by introducing space-filling amino acids at critical
positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human
5-lipoxygenase exhibited a similar specificity as the wild-type enzyme
but double, triple, and quadruple mutations led to a gradual alteration
of the positional specificity from 5S- via 8S-
toward 15S-lipoxygenation. The quadruple mutant
F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase
activity (85-95%), with
(8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.
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INTRODUCTION |
Lipoxygenases (LOXs)1
constitute a heterogeneous family of lipid-peroxidizing enzymes that
catalyze the dioxygenation of free and/or esterified polyunsaturated
fatty acids to their corresponding hydroperoxy derivatives. In mammals
LOXs are categorized with respect to their positional specificity of
arachidonic acid oxygenation into 5-, 8-, 12-, and 15-LOXs (1, 2). In
contrast, plant physiologists prefer a linoleic acid-related enzyme
nomenclature since arachidonic acid is only a minor fatty acid in
plants. Mammalian 5-LOXs are key enzymes in the biosynthesis of
leukotrienes, which are important mediators of inflammatory and
anaphylactic disorders (3, 4). During the past 10 years, 5-LOX
inhibitors and leukotriene receptor antagonists have been developed as
anti-asthmatic drugs, and some of them are now available for
prescription (5, 6). Mammalian 15-LOXs have been implicated in
peroxisome proliferation activating receptor-
-mediated cell
signaling (7), in cell development and maturation (8, 9), as well as in
the pathogenesis of atherosclerosis (10, 11). The intracellular
activity of LOXs is regulated on pre-translational, translational, and
post-translational levels. Expression of the human 5-LOX is
up-regulated by transforming growth factor (12), and melatonin
represses the 5-LOX pathway in B-lymphocytes (13). The interleukins-4
(14) and -13 (15) induce 15-LOX expression in monocyte/macrophages, and
this regulatory process involves activation of the transcription factor
STAT6 (16) as well as JAK2 and Tyk2 kinases (17). Translation of the
15-LOX mRNA is prevented during early stages of red cell maturation when special non-histone nuclear proteins (heteronuclear
ribonucleaoproteins K and E1) bind to a repetitive sequence in the
3'-untranslated region (18).
Although the positional specificity of arachidonic acid oxygenation is
decisive for mammalian LOX classification, the structural reasons for
the variation of this enzyme property remain unclear. The crystal
structures of two plant (19-21) and one mammalian LOX (22) suggest a
U-shaped hydrophobic substrate-binding pocket containing the catalytic
non-heme iron. Site-directed mutagenesis studies identified Phe-353
(23), Ile-418 and Met-419 (24), as well as Ile-593 (25) as sequence
determinants of the reticulocyte-type 15-LOXs and similar experiments
on various 12-LOX isoforms confirmed these results (26, 27). More
recently, Tyr-603 and His-604 have been identified as critical amino
acids for the positional specificity of the murine epidermis
8S- and the human epidermis-type 15S-LOX isoforms
(28). Unfortunately, neither specificity-related mutagenesis data nor
x-ray coordinates are currently available for the pharmacologically
most relevant human leukocyte 5-LOX.
For the time being, there are two hypotheses that rationalize the
mechanistic differences between arachidonic acid 5- and 15-lipoxygenation (29). (i) The "orientation-based" hypothesis suggests that, for 15-lipoxygenation, arachidonic acid may slide into
the substrate binding pocket with its methyl terminus first and may
adopt a steric configuration at the active site favoring oxygen
insertion at C-15 of the arachidonic acid backbone. In contrast, for
arachidonic acid 5-lipoxygenation, an inverse, head-to-tail substrate
orientation was assumed (30). (ii) According to the "space-based"
hypothesis, the substrate alignment at the active site is conserved
among all LOX isoforms and the volume of the substrate binding pocket
appears to be decisive for the positional specificity (31). Molecular
modeling suggested that the substrate binding cavity of 5-LOXs is about
20% bigger than that of 15-LOXs (22, 31), and the additional space may
allow an optimal substrate orientation for 5-lipoxygenation.
To test the "space-based" hypothesis, we attempted to alter the
volume of the substrate binding pocket by site-directed mutagenesis and
determined the positional specificity of the LOX mutants. Since
previous experiments failed to convert mammalian 12/15-LOXs to
5-lipoxygenating enzyme species (24, 25, 27), an inverse mutagenesis
strategy was selected. Crucial amino acids of the human leukocyte 5-LOX
were replaced with the more space-filling counterparts present in the
reticulocyte-type 15-LOXs. We found that reduction of the active site
volume by multiple site-directed mutagenesis gradually altered the
positional specificity from 5S- via 8S- toward
15S-lipoxygenation.
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MATERIALS AND METHODS |
Chemicals--
The chemicals used were from the following
sources:
(5Z,8Z,11Z,14Z)-eicosa-5,8,11,14-tetraenoic
acid (arachidonic acid),
(5S,6E,8Z,11Z,14Z)-5-hydro(pero)xyeicosa-6,8,11,14-tetraenoic acid (5S-H(p)ETE), CaCl2, EDTA, ATP, and sodium
borohydride from Serva (Heidelberg, Germany); ampicillin from Life
Technologies, Inc. (Eggenstein, Germany); dipalmitoyl
phosphatidylcholine, isopropyl-
-D-thiogalactopyranoside (IPTG), and ATP-Sepharose from Sigma-Aldrich (Deisenhofen, Germany); 12S,5Z,8Z,10E,14Z)-12-hydroxy-5,8,10,14-eicosatetraenoic
acid (12S-HETE),
(8S,5Z,9E,11Z,14Z)-8-hydroxyeicosa-5,9,11,14-tetraenoic acid (8S-HETE),
(15S,5Z,8Z,11Z,13E)-15-hydro(pero)xy-5,8,11,13-eicosatetraenoic acid (15S-H(p)ETE),
(5S,15S,6E, 8Z,11Z,13E)-5,15-dihydroxy-6,8,11,13-eicosatetraenoic acid (5S,15S-diHETE), and
(8S,15S,5Z,9E,11Z,13E)-5,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8S,15S-diHETE) from Cayman Chemical
(distributed by Alexis GmbH, Grünberg, Germany); HPLC solvents
from Merck (Darmstadt, Germany). Restriction enzymes were purchased
from New England Biolabs (Schwalbach, Germany). Phage T4 ligase,
Pwo polymerase, and sequencing kits were obtained from Roche
Molecular Biochemicals (Mannheim, Germany), and the Escherichia
coli strain HB 101 was purchased from Invitrogen (San Diego, CA).
Oligonucleotide synthesis was carried out at TiB-Molbiol (Berlin,
Germany). The human 5-LOX cDNA (cloned into the Bluescript SK+
cloning vector) was a kind gift of Dr. A. Habenicht (Heidelberg, Germany).
Bacterial Expression and Site-directed Mutagenesis--
In order
to express the human 5-LOX as non-fusion protein, its cDNA was
subcloned into the expression plasmid PKK 233-2. For this purpose we
introduced a NcoI restriction site at the starting ATG and a
HindIII site just behind the stop codon. The
NcoI/HindIII restriction fragment was then
ligated into the expression vector, and bacteria (HB 101) were
transformed with the recombinant plasmid. Site-directed mutagenesis of
the human 5-LOX was carried out by the polymerase chain
reaction-overlap extension technique using mismatching synthetic
oligonucleotides. The polymerase chain reaction products containing the
mutations were digested with appropriate restriction enzymes and
inserted into the wild-type expression plasmid. Transformed bacteria
were replated. For each mutation 20-30 clones were screened for the
expression of the mutant 5-LOX by restriction mapping and activity
assay. Several 5-LOX-positive clones were sequenced. For the final
activity assay, one sequenced clone was replated, five well separated
colonies were picked, and the bacteria were cultured at 37 °C in 5 ml of LB medium containing 0.1 mg/ml ampicillin to an optical density
at 600 nm of about 0.5. Then LOX expression was induced by addition of
IPTG (1 mM final concentration). After 12 h at
30 °C, bacteria were spun down, washed with phosphate-buffered
saline, resuspended in 0.5 ml of 0.1 M phosphate buffer, pH
7.4, containing 1 mM EDTA, and were kept on ice for 10 min.
The cells were lysed by sonication with a Labsonic U-tip sonifier
(Braun, Melsungen, Germany), cell debris was removed by centrifugation,
and the lysis supernatant was used for activity assay or further LOX purification.
Activity Assays and Enzyme Purification--
For activity
assays, those fermentation samples were selected that showed a
comparable expression level of the 5-LOX species as indicated by
immunoblotting. LOX activity was assayed in the lysis supernatants
either spectrophotometrically recording the increase in absorbance at
235 nm or by HPLC quantification of the LOX products. For HPLC
analysis, aliquots of the bacterial lysate supernatants were incubated
for 15 min at room temperature with 0.1 mM arachidonic acid
in the presence of 0.4 mM CaCl2, 40 µg/ml
dipalmitoyl phosphatidylcholine, and 0.1 mM ATP (final reaction volume of 0.5 ml). The hydroperoxy compounds formed were reduced with sodium borohydride to the corresponding hydroxy
derivatives, the mixture was acidified to pH 3, and 0.5 ml of ice-cold
methanol was added. The protein precipitate was spun down, and aliquots of the clear supernatant were injected directly for quantification of
the LOX products to RP-HPLC. Activity assays of the purified enzyme
preparations (see below) were carried out spectrophotometrically or by
HPLC quantification of the LOX products. For the spectrophotometric measurements, the assay mixture was a 0.1 M
sodium/potassium phosphate buffer, pH 7.4, containing 0.4 mM CaCl2, 0.1 mM EDTA, 0.1 mM ATP, 12 µg|ml dipalmitoyl phosphatidylcholine, and
0.1 mM arachidonic acid as substrate. To 1 ml of this
mixture aliquots (50-200 µl) of the purified enzyme preparations
were added, and the increase in absorbance at 235 nm was recorded at
room temperature.
Selected mutants of the 5-LOX were purified by FPLC on a
semipreparative MonoQ column (Amersham Pharmacia Biotech, Uppsala, Sweden) or by ATP affinity chromatography on an open bed ATP-Sepharose column (32). For this purpose, a LOX-active clone was picked with a
sterilized toothpick and 10 ml of LB medium containing ampicillin (0.1 mg/liter) were inoculated. After overnight culture at 37 °C, 1 ml of
this pre-culture was added to 200 ml of LB medium (0.1 mg/liter
ampicillin) and the bacteria were grown at 37 °C to an optical
density at 600 nm of about 0.5. Expression of the recombinant LOX
species was then induced with 1 mM IPTG, and the culture
was incubated for additional 12 h at 30 °C. Cells were spun
down, washed with phosphate-buffered saline, and resuspended in 2 ml of
50 mM triethanolamine/HCl buffer, pH 7.3, containing 2 mM EDTA and 10 mM mercaptoethanol. Cells were
lysed by sonication (three times for 20 s) with a Labsonic U-tip
sonifier (Braun), and cell debris was removed by centrifugation. For
purification of the LOX species, aliquots of the supernatant were
injected into FPLC and the chromatograms were developed with a linearly increasing sodium chloride gradient. Alternatively, aliquots of the
supernatant were applied to an ATP-Sepharose column (gel volume of 3 ml). This column was washed with 10 ml of 50 mM
triethanolamine/HCl buffer, pH 7.3, containing 2 mM EDTA,
10 mM mercaptoethanol, and 1 M NaCl to remove
unspecifically bound proteins, and the 5-LOX was then eluted with the
same buffer containing 100 mM NaCl and 15 mM
ATP. Fractions of 2 ml were collected, and the LOX activity was
assayed. With these one-step purification procedures, the enzyme was
purified about 500-fold, but we did not reach electrophoretic homogeneity (10-30% purity of the final enzyme preparation). Attempts to further purify the mutants by various FPLC techniques failed, since
we experienced severe losses in enzyme activity. In separate experiments we compared the positional specificity of crude and purified enzyme preparations and did not observe significant differences.
HPLC of Oxygenated Fatty Acids--
HPLC was performed on a
Shimadzu system connected to a Hewlett Packard diode array detector
1040. Reverse phase-HPLC was carried out on a Nucleosil C-18 column
(Macherey-Nagel, KS system, 250 × 4 mm, 5-µm particle size)
coupled with an appropriate guard column (30 × 4 mm, 5-µm
particle size). For analysis of the mono-oxygenated fatty acids (HETE
and HpETE isomers), a solvent system of methanol/water/acetic acid
(80/20/0.1, by volume) was used at a flow rate of 1 ml/min. For the
diHETE derivatives, the water content of the solvent system was
somewhat increased (methanol/water/acetic acid, 75/25/0.1, by volume)
The chromatographic scale was calibrated for conjugated dienes by
injecting known amounts of 15-HETE and for conjugated trienes using
8S,15S-diHETE as reference. Straight phase-HPLC (SP-HPLC) was performed on a Zorbax-SIL column (250 × 4.6 mm, 5-µm particle size) using a solvent system of
n-hexane/2-propanol/acetic acid (100/2/0.1, by volume) at a
flow rate of 1 ml/min. For enantiomer separation of the different
hydroxy fatty acid isomers, chiral phase HPLC was performed under the
following conditions: for 15R/S-HETE, analysis of
the free acid on a Chiralcel OD column (250 × 4 mm, 5-µm
particle size) with the solvent system
n-hexane/2-propanol/acetic acid (100/5/0.1, by volume) and a
flow rate of 1 ml/min; for 8R/S-HETE, analysis of
the methyl ester on a Chiralcel OD column (250 × 4 mm, 5-µm
particle size) with the solvent system
n-hexane/2-propanol/acetic acid (100/4/0.1, by volume) and a
flow rate of 1 ml/min; for 5R/S-HETE, analysis of
the methyl ester on a Chiralcel OB column (250 × 4 mm, 5-µm
particle size) with the solvent system
n-hexane/2-propanol/acetic acid (100/5/0.1, by volume) and a
flow rate of 1 ml/min.
Miscellaneous Methods--
For basic kinetic characterization of
the 5-LOX species, the oxygenation of arachidonic acid was quantified
either spectrophotometrically or by RP-HPLC quantification of the LOX
products. Km-values were determined varying the
substrate concentration in the range of 10-120 µM, and
the linear part of the Lineweaver-Burk plot was evaluated. HPLC
standards of racemic 5R/S-HETE,
8R/S-HETE, 12R/S-HETE, and
15R/S-HETE were prepared by vitamin E-controlled autooxidation of arachidonic acid methyl ester and subsequent HPLC
separation of the positional isomers. The free hydroxy fatty acids were
obtained by alkaline hydrolysis of the methyl esters and subsequent
reverse phase HPLC purification. For immunoblotting the bacteria were
lysed as described for the activity assays. The cell debris was spun
down, and aliquots of the lysis supernatants were applied to SDS-gel
electrophoresis. The proteins were transferred to a nitrocellulose
membrane by a semidry blotting procedure, and the blots were probed
with a polyclonal antibody against the human 5-LOX (kind gift of A. Habenicht, Heidelberg, Germany). Anaerobiosis was achieved by repeated
securation of the assay mixture and subsequent flushing with argon.
Hydroxy fatty acids were methylated with diazomethane in diethylether,
and the resulting methyl esters were purified by RP-HPLC.
 |
RESULTS |
Bacterial Expression of the Human 5-LOX and Characterization of the
Recombinant Enzyme--
Bacteria transformed with the recombinant
expression plasmid containing the human 5-LOX cDNA express the
functional enzyme as indicated by activity assays (Fig.
1) and immunoblot analysis (data not
shown). An arachidonic acid oxygenase activity of 0.5 ± 0.1 µg
of 5-HpETE formation/15-min incubation period (n = 27) was measured per milliliter of culture fluid. For more comprehensive characterization the recombinant enzyme was expressed in a 200-ml liquid culture and purified by affinity chromatography on
ATP-Sepharose. The purified enzyme exhibited similar enzymatic
characteristics (kinetic lag-phase, substrate specificity, suicidal
inactivation) as crude enzyme preparations (bacterial lysis
supernatant) and as the native enzyme prepared from human
leukocytes.

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Fig. 1.
Product pattern formed by the purified
recombinant human 5-LOX. A 200-ml fermentation of the human 5-LOX
was performed, and the recombinant enzyme was purified by affinity
chromatography on ATP-Sepharose. The active fractions were pooled and
the product pattern of arachidonic acid oxygenation was analyzed by
RP-HPLC as described under "Materials and Methods." The chemical
structure of the major product was confirmed by coinjection with
authentic standards in RP- and SP-HPLC and by UV spectroscopy
(inset). The S/R ratio (9:1) of the 5-HpETE
formed was determined by chiral phase HPLC of the hydroxy fatty acid
methyl ester obtained after suitable derivatization (reduction and
methylation).
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Single Point Mutations of Sequence
Determinants--
Structure-based sequence alignment of mammalian 5- and 15-LOXs (Fig. 2) suggested potential
sequence determinants for the positional specificity of the human
5-LOX, and these residues were targeted by site-directed mutagenesis.
In order to decrease the volume of the substrate-binding pocket, we
mutated these amino acids to the more space-filling counterparts
present in reticulocyte-type 15-LOXs. Single point mutations at
positions 424 (A424I) or 425 (N425M) led to enzyme species forming
significant amounts of 8S-HpETE in addition to
5S-HpETE, as indicated by HPLC analysis of the corresponding
hydroxy derivatives obtained after borohydride reduction of the primary
oxygenation products (Fig. 3). In
contrast, A603I and C561F exchange did not influence the positional
specificity (Table I). Phe-353, which
constitutes a sequence determinant of mammalian 12/15-LOXs, aligns with
Phe-359 of the human 5-LOX. In order to reduce the volume of the active
site, we mutated Phe-359 to a more bulky Trp. From Table I it can be
seen that the F359W mutant exhibited an altered positional specificity
when compared with the wild-type enzyme. Although 5S-HpETE
was still the major oxygenation product, 8S-HpETE
contributed about 30% to the product mixture. Here again the
corresponding hydroxy fatty acids obtained after borohydride reduction
of the peroxy fatty acids formed were analyzed. The F359H mutant formed
little more 8S-HpETE than the wild-type control, but
significantly less than the F359W mutant (Table
II). These minor alterations may be
explained by the fact that a Phe-His exchange may not significantly
alter the volume of the substrate binding pocket since both amino acids
are of similar size (33). When smaller site chains were introduced at
this position (F359L, F359V), inactive enzyme species resulted (Table
II). It may be speculated that enlargement of the active site leads to
a sloppy substrate binding and, thus, to a strongly reduced enzymatic
activity.

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Fig. 2.
Amino acid alignment of mammalian 5- and
15-LOXs. The one-letter code for amino acids
is used. The numbers indicate the position of the amino acid
for the human 5-LOX.
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Fig. 3.
Single point mutation of sequence
determinants alters the positional specificity of the human 5-LOX.
For the wild-type human 5-LOX and for each of the mutant enzyme
species, a 5-ml culture was grown overnight and the LOX activity was
assayed in the lysis supernatant as described under "Materials and
Methods." Arachidonic acid oxygenation products extracted under
reducing conditions were quantified by RP-HPLC. The y axis
for each sample was scaled for the 5-HETE peak. Chiral phase HPLC of
the reduced and methylated reaction products indicated a strong
preponderance of the S-isomers.
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Table I
Positional specificity of various 5-LOX single mutants
The LOX activity is expressed as micrograms of HETE formation/ml of
fermentation culture during a 15-min incubation period. Product
formation was quantified by RP-HPLC analyzing the HETEs obtained after
borohydride reduction of the HpETEs formed. The activity data represent
the means of five independent measurements with different LOX-positive
clones.
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Table II
Positional specificity of human 5-LOX mutated at position 359
The LOX activity is expressed as micrograms of HETE formation/ml of
fermentation culture during a 15-min incubation period. Product
formation was quantified by RP-HPLC analyzing the HETEs obtained after
borohydride reduction of the HpETEs formed. The activity data represent
the means of five independent measurements with different LOX-positive
clones. ND, not determined.
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Taken together these results indicate that Phe-359, Ala-424, and
Asn-425 may be considered as sequence determinants for the positional
specificity of the human 5-LOX and that introduction of more
space-filling residues at these positions leads to an increased share
of 8S-HpETE formation. Unfortunately, these alterations were
only partial, and we did not observe any chiral products of
15-lipoxygenation.
Multiple Point Mutations of the Sequence Determinants--
To
further reduce the volume of the substrate-binding pocket, we combined
the effective single mutations listed in Table I. When N425M was
combined with F359W and A424I (F359W/N425M, A424I/N425M double
mutants), the share of 8S-HpETE formation was strongly augmented (Table III). Here again, we did
not observe significant amounts of chiral 15-HETE when analyzing the
product mixture after borohydride reduction. After combining the F359W
exchange with an A424I mutation, we no longer observed a major 5-HpETE
formation. Instead, 8S-HpETE was identified as main
oxygenation product (Table III). Interestingly, this double mutant
converted arachidonic acid consistently to a small (5%) but
significant share of chiral 15S-HpETE, as indicated by HPLC
analysis of the hydroxy fatty acids obtained after borohydride
reduction. This effect was even more pronounced when the
F359W/A424I/N425M triple mutant was constructed. As indicated in Fig.
4, this mutant enzyme converted
arachidonic acid to an almost 1:1 mixture of 8S- and
15S-HpETE. Although the rate of arachidonic acid oxygenation
was lower than that of the wild-type controls (0.1 µg of HpETE/ml of
culture fluid culture versus 0.58 µg/ml culture fluid of
the wild-type enzyme), product formation was completely
enzyme-controlled as indicated by the high degree of chirality of the
hydroperoxy fatty acids formed (Fig. 4, insets).
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Table III
Positional specificity of multiple point mutants of the human 5-LOX
The LOX activity was expressed as micrograms of HETE formation/ml of
fermentation culture during a 15-min incubation period. Product
formation was quantified by RP-HPLC analyzing the HETEs obtained after
borohydride reduction of the HpETEs formed. The activity data represent
the means of five independent measurements with different LOX-positive
clones.
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Fig. 4.
Arachidonic acid 15-lipoxygenation by the
F359W/A424I/N425M 5-LOX triple mutant. The wild type and mutant
5-LOX species were expressed in E. coli (1 liter of culture)
and purified by FPLC on a Mono Q-Sepharose column. LOX-containing
fractions were pooled, activity assays were carried out, and the
oxygenation products extracted under reducing conditions were analyzed
by RP-HPLC. Insets, analysis of the enantiomer composition
of 15- and 8-HETE obtained after suitable derivatization (reduction and
methylation) of the primary oxygenation products.
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It has been reported before that I593A exchange altered the positional
specificity of the rabbit 15-LOX (25), but the inverse strategy with
the human 5-LOX (A603I) was not successful (Table I). It may be
speculated that reduction of the active site volume achieved by this
mutation was not sufficient to alter the enzyme specificity. However,
when A603I exchange was carried out with the F359W/A424I/N425M triple
mutant, a LOX species was created, which exhibited a major
15S-LOX activity (Fig. 5). We
found that the F359W/A424I/N425M/A603I quadruple mutant exhibited an
impaired oxygenase activity (mutant: 0.16 ± 0.08 µg of HETE/ml
of culture fluid, n = 20; wild-type control: 0.58 µg
of HETE/ml of culture fluid, n = 27) and that the share
of 15S-HpETE formation varied between 75% and 90%. Here
again, the high degree of chirality (Fig. 5, insets) of the
two major reaction products indicated that the dioxygenase reaction was
completely enzyme-controlled.

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Fig. 5.
The F359W/A424I/N425M/A603I 5-LOX quadruple
mutant is a major 15-LOX. The F359W/A424I/N425M/A603I 5-LOX mutant
was expressed in E. coli as described under "Materials and
Methods." The enzyme was purified by affinity chromatography on
ATP-Sepharose, the LOX active fractions were pooled, and aliquots were
used for activity assays (0.5-ml assay volume) as described under
"Materials and Methods." After 15 min of incubation at room
temperature, the hydroperoxy fatty acids formed were reduced with
sodium borohydride, the sample was acidified to pH 3, and 0.5 ml of
ice-cold methanol was added. Protein precipitate was spun down, and the
entire assay mixture was injected for RP-HPLC purification of the
hydroxy fatty acids. The HETE fraction was collected, the solvent was
evaporated, the residue was reconstituted in hexane, and aliquots were
analyzed by SP-HPLC. Inset, chiral-phase HPLC of the 15- and
8-HETE prepared by SP-HPLC.
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Multiple point mutations are always dangerous since the
three-dimensional structure may be altered at different sites, and, thus, the enzyme may lose activity. In fact, our triple and quadruple mutants exhibited reduced arachidonic acid oxygenase activities. In
order to avoid multiple mutations but to obtain comparable spatial
effects, we introduced more bulky amino acids at the positions of the
sequence determinants (Table IV). A424F
and N425F exchange led to enzyme species with multiple positional
specificity, and we detected variable amounts of 15S-HETE
after borohydride reduction of the hydroperoxy fatty acids formed.
Unfortunately, neither the single mutants A603F and A603W nor the
double mutant A424F/N425F were catalytically active.
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Table IV
Phenylalanine scan of sequence determinants
The LOX activity was expressed as micrograms of HETE formation/ml of
fermentation culture during a 15-min incubation period. Product
formation was quantified by RP-HPLC analyzing the HETEs obtained after
borohydride reduction of the HpETEs formed. The activity data represent
the means of five independent measurements with different LOX-positive
clones. ND, not determined.
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Mechanistic Studies--
In order to exclude that multiple
mutation may have converted the human 5-LOX to an arachidonate
15-hydroxylase, we analyzed the primary oxygenation products prepared
under non-reducing conditions. Since hydroperoxy fatty acids are not
well separated in RP-HPLC from the corresponding hydroxy compounds, the
oxygenated fatty acids were prepared by RP-HPLC and further analyzed by
SP-HPLC. From Fig. 6 (panel
A), it can be seen that the wild-type 5-LOX converts
arachidonic acid mainly to 5-HpETE. Using the F359W/A424I/N425M/A603I mutant as catalyst, the major oxygenation product co-eluted with an
authentic standard of 15-HpETE (panel B). When
product preparation was carried out under reducing conditions, we
detected the corresponding hydroxy derivatives (Fig. 6), and these data
confirm that fatty acid hydroperoxides constitute the primary reaction
products. Under anaerobic conditions, arachidonic acid oxygenation was
prevented, and experiments with H218O did not
show any incorporation of heavy oxygen isotopes. These data indicate
that the quadruple 5-LOX mutant remains a true LOX and that multiple
mutations do not convert the LOX to a fatty acid hydroxylase.

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Fig. 6.
Formation of hydroperoxy fatty acids by the
wild type 5-LOX and its F359W/A424I/N425M/A603I quadruple mutant.
The wild type 5-LOX and its F359W/A424I/N425M/A603I mutant were
expressed in E. coli as described under "Materials and
Methods." The enzyme species were purified by affinity chromatography
on ATP-Sepharose, the LOX active fractions were pooled, and aliquots
were used for activity assays (0.5-ml assay volume). After 15 min of
incubation at room temperature (no borohydride treatment), 0.5 ml of
ice-cold acidic methanol (1% acetic acid) was added, protein
precipitate was spun down, and the entire assay mixture was injected
for RP-HPLC preparation. The fractions containing the oxygenated fatty
acids were collected, the solvent was evaporated, the remaining lipids
were reconstituted in hexane, and aliquots were injected for SP-HPLC
analysis. The arrows above the traces indicate
the retention times of the authentic standards. A, wild type
5-LOX; B, F359W/A424I/N425M/A603I mutant.
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Arachidonic acid methyl ester was not a suitable substrate for the wild
type 5-LOX and its F359W/A424I/N425M/A603I mutant. This finding was not
surprising for the wild-type enzyme since similar data have been
reported before for the native enzyme (34). However, the
15-lipoxygenating quadruple mutant was expected to oxygenate methyl
arachidonate since other 15-lipoxygenating enzyme species
(reticulocyte-type 15-LOX) accept fatty acid methyl esters as
substrate. Our finding that this 5-LOX mutant is not capable of
oxygenating arachidonic acid methyl ester suggests that the enzyme
species exhibits a substrate specificity, which is more closely related
to the wild-type 5-LOX and/or to the epidermis-type 15-LOX (28).
It has been reported before that the positional specificity of certain
LOX isoforms strongly depends on the pH of the reaction mixture (35).
To exclude that the differences in the positional specificity of the
5-LOX mutants may be due to a different pH sensitivity, we investigated
the pH dependence of both the wild type 5-LOX and its 15-lipoxygenating
quadruple mutant. As shown in Fig. 7, the
activity of both enzyme species showed somewhat different pH profiles.
For the wild type 5-LOX, a pH optimum of 8.0 was determined under our
experimental conditions and 5-HpETE turned out to be the exclusive
oxygenation product over the entire pH range. The
F359W/A424I/N425M/A603I quadruple mutant oxygenated arachidonic acid
most effectively at pH 8.5, and 15-HpETE was identified as major
reaction product at all pH values tested.

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Fig. 7.
pH profiles of the LOX activity of the wild
type 5-LOX and its F359W/A424I/N425M/A603I mutant. Enzyme
preparation was carried out as described in the legend to Fig. 6. The
different pH values were adjusted in a phosphate/borate buffer mixture
by the addition of 1 N HCl or 1 N NaOH. The
relative LOX activity was determined by quantifying the formation of
hydroxy fatty acids (after borohydride reduction) by RP-HPLC after a
15-min incubation period. No major alterations in the positional
specificity were observed during the entire pH range.
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Basic kinetic characterization of the wild-type 5-LOX, its
8-lipoxygenating double mutant, and its 15-lipoxygenating quadruple mutant revealed that the mutant isoforms exhibit a somewhat reduced substrate affinity when compared with the wild-type enzyme
(Km of 63 µM for the F359W/A424I
double mutant, 57 µM for the F359W/A424I/N425M/I603A quadruple mutant, and 35 µM for the wild-type 5-LOX).
However, the differences observed were not very dramatic.
Using different hydroxy fatty acids as substrate, we investigated the
question of whether the various 5-LOX species are capable of catalyzing
hydrogen abstraction from different bisallylic methylenes. We found
that the wild type 5-LOX strongly favors C-7 hydrogen abstraction.
5S-HETE, which lacks a C-7 bisallylic methylene, was not
oxygenated by this enzyme. In contrast, 12S-HETE and
15S-HETE containing C-7 bisallylic methylenes were
effectively oxygenated at C-5 (Table V).
The 15-lipoxygenating quadruple mutant was less restrictive with
respect to the site of hydrogen abstraction. 5S-HETE, which
contains two bisallylic methylenes (C-13 and C-10), was oxygenated at
C-15 and at C-12, indicating that the mutant enzyme can catalyze
hydrogen abstraction from both bisallylic methylenes to an almost
similar extent (Table V). 15S-HETE, which lacks the C-13
bisallylic methylene, was oxygenated at C-8, and this reaction involves
a C-10 hydrogen removal. Interestingly, the reaction rate of
15S-HETE oxygenation was more than 1 order of magnitude
lower than that of 5S-HETE conversion. 12S-HETE, which only contains a C-7 bisallylic methylene, was not oxygenated by
the F359W/A424I/N425M/A603I mutant. This result is quite plausible since the quadruple mutant was not capable of catalyzing C-7 hydrogen abstraction from arachidonic acid (Fig. 5).
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Table V
Oxygenation of various HETE isomers by the wild-type human 5-LOX and
its 15-lipoxygenating quadruple mutant
The enzyme species were prepared as described in the legend to Fig. 6.
Aliquots of the pooled LOX-active fraction of the ATP-Sepharose
affinity chromatography were incubated in the standard assay system
using different hydroxy fatty acids as substrate (20 µM
final concentration, 0.5 ml of assay sample). After borohydride
reduction the incubation mixture was acidified, acidic methanol (1%
acetic acid) was added to reach a final concentration of 50%, and the
entire mixture was injected for RP-HPLC analysis of the dihydroxy fatty
acids (see "Materials and Methods"). The chemical structure of the
various diHETE isomers was confirmed by UV spectroscopy and coinjection
with authentic standards.
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 |
DISCUSSION |
For mammalian 12- and 15-LOXs the amino acids, which align with
the Phe-353 (23), Ile-418, Met-419 (24), and Ile-593 (25) of the rabbit
reticulocyte 15-LOX, have been identified as sequence determinants for
the positional specificity. However, the question of whether these
amino acids may also be important for the specificity of the
pharmacologically most relevant mammalian 5-LOXs has not been
investigated so far. Since previous site-directed mutagenesis studies
and chimera formation failed to convert mammalian 12- and 15-LOXs to
5-lipoxygenating enzyme species (24, 25, 27), we followed an inverse
strategy and mutated critical amino acids of the human 5-LOX to the
more space-filling counterparts present in mammalian 15-LOXs. Our data
suggest that Phe-359, Ala-424, Asn-425, and Ala-603 of the human 5-LOX
are involved in positioning substrate fatty acids at the active site
and, thus, may be considered as sequence determinants for the
positional specificity. When these amino acids alone or in combination
with each other were mutated to more space-filling residues, we
observed a gradual conversion of the human 5-LOX to 8S- and
further to 15S-lipoxygenating enzyme species. These results,
which indicate the principle possibility of interconverting 5- and
15-lipoxygenating enzyme species, are rather surprising since the
stereochemistry of 5S- and 15S-lipoxygenation is
quite different and both isoenzyme classes do not share a high degree
of amino acid identity (64% between human 5-LOX and reticulocyte-type 15-LOX).
In order to find out whether the effects observed are peculiar for the
human leukocyte 5-LOX, we carried out mutagenesis studies on a potato
tuber 5-LOX (36). The recombinant wild-type enzyme oxygenated
arachidonic acid to a mixture of 5- and 8-HpETE, as indicated by
RP-HPLC of the corresponding hydroxy compounds obtained after
borohydride reduction (5-HpETE/8-HpETE ratio of 65:35). No 15-HpETE
formation was observed. Val-580 of this enzyme, which aligns with
Asn-425 of the human 5-LOX, was mutated to a more space-filling His,
and the resulting V580H mutant converted arachidonic acid to a 60:20:20
mixture of 5-, 8-, and 15-HpETE (data not shown). With this enzyme, a
single point mutation altered the positional specificity in favor of
15S-lipoxygenation. However, the alterations were only
partial and we did not carry out multiple mutations on this plant
LOX.
The x-ray data of the rabbit 15-LOX (22) and more recent mutagenesis
studies on this enzyme (25) suggested that the bottom of the
substrate-binding pocket is defined by the side chains of Phe-353,
Ile-418, and Ile-593. The walls of the cavity are lined by hydrophobic
residues, but in the proximity of the iron center there are several
polar amino acids. The opening of the substrate-binding cleft may be
capped by Arg-403, which was modeled to interact with the substrates'
carboxylate (22, 37). Arg-403 of the rabbit enzyme aligns with Lys-409
of the human 5-LOX (Fig. 2), and we mutated this lysine to a positively
charged arginine (K409R) and to an apolar leucine (K409L).
Surprisingly, we did not observe major differences when the positional
specificity and the specific activities of the wild type and mutant
enzyme species were compared. These data suggest that Lys-409 of the human 5-LOX may not be of major importance for enzyme/substrate interaction.
In mammals 5- and 15-LOXs may have different biological functions.
Thus, unspecific LOX inhibitors, which have no preference for a
particular LOX isoform, may not be developed as potential drugs,
because of probable unwanted side effects. However, the observation
that the volume of the active site of mammalian 5-LOXs is bigger than
that of other isoforms may be useful for the development of specific
5-LOX inhibitors. The mutant enzyme species created in this study may
be used in molecular test systems searching for isoform-specific LOX inhibitors.
 |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant Ku 961/7-1 and European Commission Grant BMH-98-3191.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-30-20937539; Fax: 49-30-20937300; E-mail:
hartmut.kuehn@charite.de.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M005114200
 |
ABBREVIATIONS |
The abbreviations used are:
LOX, lipoxygenase;
5S-H(p)ETE, (5S,6E,8Z,11Z,14Z)-5-hydro(pero)xyeicosa-6,8,11,14-tetraenoic
acid;
15S-H(p)ETE, (15S,5Z,8Z,11Z,13E)-15-hydro(pero)xyeicosa-5,8,11,13-tetraenoic
acid;
12S-H(p)ETE, (12S,5Z,8Z,10E,14Z)-12-hydro(pero)xyeicosa-5,8,10,14-tetraenoic
acid;
8S-H(p)ETE, (8S,5Z,9E,11Z,14Z)-8-hydro(pero)xyeicosa-5,9,11,14-tetraenoic
acid;
FPLC, fast protein liquid chromatography;
IPTG, isopropyl-
-D-thiogalactopyranoside;
5S, 15S-diHETE,
(5S,15S,6E,8Z,11Z,13E)-5,15-dihydroxy-6,8,11,13-eicosatetraenoic
acid;
8S, 15S-diHETE,
(8S,15S,5Z,9E,11Z,13E)-8,15-dihydroxy-5,9,11,13-eicosatetraenoic
acid;
5S, 12S-diHETE,
(5S, 12S,6E,8Z,10E,14Z)-5,15-dihydroxy-6,8,10,14-eicosatetraenoic
acid;
RP(SP)-HPLC, reverse (straight) phase high performance liquid
chromatography;
HPLC, high performance liquid chromatography;
H(p)ETE, fatty acid hydro(pero)xide;
diHETE, dihydroxy fatty
acid.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.