Cloning of the manganese lipoxygenase gene reveals homology
with the lipoxygenase gene family
Lena Ho¨ rnsten
1
, Chao Su
1
, Anne E. Osbourn
2
, Ulf Hellman
3
and Ernst H. Oliw
1
1
Department of Pharmaceutical Biosciences, Uppsala Biomedical Centre, Uppsala, Sweden;
2
The Sainsbury Laboratory,
John Innes Centre, Norwich, UK;
3
Ludwig Institute for Cancer Research, Uppsala Biomedical Centre, Uppsala, Sweden
Manganese lipoxygenase was isolated to homogeneity from
the take-all fungus, Gaeumannomyces graminis. The C-ter-
minal amino acids and several internal peptides were
sequenced, and the information was used to obtain a cDNA
probe by RT/PCR. Screening of a genomic library of
G. graminis yielded a full-length clone of the Mn-Lipoxyg-
enase gene. cDNA analysis showed that the gene spanned
2.6 kb and contained one intron (133 bp). Northern blot
analyses indicated two transcripts (2.7 and 3.1 kb). The
deduced amino-acid sequence of the Mn-Lipoxygenase
precursor (618 amino acids, 67.7 kDa) could be aligned with
mammalian and plant lipoxygenases with 23–28% identity

Mn-Lipoxygenase belongs to the lipoxygenase gene family
and that its unique biochemical properties might be related
to structural differences in the metal centre and a helix 9 of
lipoxygenases rather than to the metal ligands.
Keywords: ascomycete; dioxygenase; lipoxygenase; hydro-
peroxide; metalloenzyme.
Lipoxygenases (LOX; EC 1.13.11.12) are widely distributed
in mammals and plants and oxygenate polyunsaturated
fatty acids to cis–trans conjugated hydroperoxides [1]. LOX
have three important biological functions. The hydroperoxy
fatty acids may act as signal molecules, either directly or
after conversion to a large variety of biologically active
products such as leukotrienes in man [2] and jasmonic acid
in plants [3]. LOX can also catalyze physiological break-
down of cellular membranes and organelles in the lens and
in the reticulocyte [1,4]. Plant LOX genes are activated in
response to wounding and pathogen attack [5], and reduced
plant LOX activity results in an increased susceptibility to
insects and fungal pathogens [6,7].
All LOX belong to the same gene family [1]. The pair-
wise amino-acid sequence identity of plant and animal
LOX is only 21–27%, whereas the corresponding figures
within pairs of plant or pairs of animal LOX are often
40% or higher. LOX in animals and plants contain
mononuclear nonheme Fe as the catalytic metal, which
has been demonstrated by atomic absorption spectroscopy
for soybean LOX [8], rabbit reticulocyte 15-LOX [9] and
human 5-LOX [10]. X-ray crystallography of soybean
LOX L1 and L3 [11–14], and rabbit reticulocyte 15-LOX
[15] has identified the Fe(II) ligands. These are one water

Abbreviations: LOX, lipoxygenase(s); Gga, G. graminis var avenae;
Ggt, G. graminis var tritici; Mn-LOX, manganese lipoxygenase.
Enzymes: lipoxygenases (EC 1.13.11.12).
Note: The sequences reported in this paper have been deposited in
GenBank under accession nos AY040824 and AY040825.
(Received 4 March 2002, accepted 17 April 2002)
Eur. J. Biochem. 269, 2690–2697 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02936.x
Third, Mn-LOX is the first LOX known to be secreted
by a microorganism, and it is also remarkably stable [19].
The biological function of Mn-LOX is unknown, but the
enzyme may cause oxidative damage and contribute to the
pathogenicity of G. graminis.
Analysis of the metal cofactor of Mn-LOX during
catalysis revealed important similarities with LOX. The
mononuclear metal center of Mn-LOX redox cycles
between Mn(II) in the resting state and Mn(III) in the
active state [21], whereas the metal centre of LOX redox
cycles in the same way between Fe(II) and Fe(III) [1]. The
active forms of both enzymes abstract, with stereo-speci-
ficity, a bisallylic hydrogen from their fatty acid substrates
and form a substrate radical. The free radical reacts with
molecular oxygen in a controlled fashion relative to the
hydrogen abstraction so that antarafacial oxygen insertion
is catalyzed by LOX and suprafacial oxygen insertion by
Mn-LOX [1,20].
The metal ligands contribute to the large diversity of
nonheme Fe(II) enzymes [17]. Some enzymes occur in
homologous forms with Fe or Mn as catalytic metals, and
the metal ligands can be conserved. The extradiol-cleaving
catecholdioxygenase(3,4-dihydroxyphenylacetate2,3-dioxy-

and var tritici (Ggt)] were obtained and grown as described
[19,27,28]. Qiagen plant DNeasy mini, RNeasy mini and
QIAquick gel extraction kits were from Merck Eurolab
(Stockholm, Sweden). Degenerate primers for PCR were
obtained from TIB Molbiol (Berlin, Germany), and
sequencing primers were from CyberGene (Huddinge,
Sweden). 5¢-RACE and reverse transcription of total
RNA were performed with a kit (5¢-RACE system for
rapid amplification of cDNA ends) from Life Technologies,
who also provided RNA (0.24–9.5-kb) and DNA ladders
(1-kb). Cycle sequencing kits were: Thermo Sequenase for
radiolabeled ddNTPs from Amersham Pharmacia Biotec;
and ABI Prism Big-Dye terminator from PerkinElmer.
Equipment for protein purification was as described
previously [19]. Endoglycosidase F/N-glycosidase F and
O-glycosidase were from Boehringer-Mannheim. Polyvinyl-
difluoride membranes (ProBlott) were from Applied Bio-
systems.
Purification
Mn-LOX was isolated from Ggt and Gga, and purified by
chromatography as described before [19,21]. We purified the
enzyme from two sources, as the genomic library was
obtained from Gga and internal peptides were from
Mn-LOX of Ggt. Enzymatic deglycosylation was
performed as described previously [19].
Total amino-acid composition
The peak fraction of Mn-LOX-Ggt from the gel filtration
column was analyzed directly for total amino acids [21],
whereas an additional step was used for Mn-LOX-Gga.
After gel filtration, this enzyme was purified by SDS/PAGE

The PCR (50 lL) contained 0.4 l
M
each primer, 10 m
M
Tris/HCl pH 8.3, 50 m
M
KCl, 3.0 m
M
MgCl
2
,0.2m
M
dNTPs and 1.5 U Taq DNA polymerase. The PCR
protocol was: 94 °C for 3 min, 1 cycle, followed by
94 °C for 45 s, 48 °C for 45 s, 72 °Cfor1minfor30
cycles, a final extension step (72 °C, 10 min) and then
cooling to 8 °C. The amplicons were cloned into the TA
vector pCR2.1-TOPO and used for heat shock transfor-
mation of Escherichia coli (TOP10, Invitrogen). Sequencing
was performed by the cycle sequencing method.
Ó FEBS 2002 Cloning of manganese lipoxygenase (Eur. J. Biochem. 269) 2691
Genomic library screening
The genomic library of Gga was constructed by partial
digestion of genomic DNA with TspeI and ligated into the
EcoR1 site of k-ZAP II (Stratagene) as described previously
[27]. A cDNA probe (0.33 kb) was generated by RT/PCR
using primers MnS2 and MnS1 and labeled with
32
Pusing
the random priming method [32]. Hybridization screening

(; [33]) was used for database
search and for pair wise alignments, whereas the
LASERGENE
MEGALIGN
program (Dnastar, Madison, WI, USA) was
used for multiple alignments.
RESULTS
Amino-acid analyses and degenerate oligonucleotides
Native Mn-LOX-Gga was purified to homogeneity and had
an apparent molecular size of 90–110 kDa on SDS/PAGE,
whereas Mn-LOX-Ggt appeared to be larger (100–
140 kDa) [19]. After N- and O-linked deglycosylation,
SDS/PAGE of Mn-LOX showed two bands of  67 and
 73 kDa. Mn-LOX-Gga yielded mainly the 67 kDa pro-
tein, whereas Mn-LOX-Ggt yielded both with equal inten-
sity, possibly due to incomplete deglycosylation [34]. The
total amino-acid compositions of Mn-LOX-Ggt and
Mn-LOX-Gga and of the deduced precursor proteins are
summarized in Table 1.
The four C-terminal amino acids were determined by
C-terminal sequencing as FLSV. In situ digestion of Mn-LOX-
Ggt with endoproteinase Lys-C, V8 and trypsin followed by
peptide separation and amino-acid sequencing [30] yielded
10 relatively long internal peptide sequences (including the
C-terminal peptide of 23 amino acids). Two peptides were
successfully used for design of degenerate oligonucleotide
primers: peptide-1, LYTPQPGRYAAACQGLFYLDARS
NQFLPLAIK (obtained with Lys-C) was used to design the
sense primer Mn60 (5¢-AACCAGTTCCTSCCSCTCGCS
ATCAA-3¢) and the antisense primer Mn15R (5¢-GTCGA

LOX [35], in one of the reading frames, whereas the primers
EO3A and Mn15R generated a band of 220 bp. Misprim-
ing of the EO3A primer formed the latter, as a sense primer
from this sequence (MnS2: 5¢-CCGTTCAGCGTCGAGA
GCAAGG-3¢) and an antisense primer from the other
sequence (MnS1, 5¢-TCTCGGGGATCGTGTGGAAGA
GCA-3¢) amplified a fragment of 337 bp. The latter
contained WLLAK and the amino-acid sequence of pep-
tide 1 in one of the reading frames. This amplicon was used
as a probe for screening of a genomic library of Gga and for
Northern blot analysis.
Isolation of genomic clones
About 100 000 plaques were screened with the cDNA probe
and 11 positive clones were obtained. Positive plaques were
subject to three rounds of plaque purification. All rescued
Bluescript SK phagemids seemed to contain the same insert
of  8 kb as judged from restriction enzyme analysis.
Organization of the Mn-LOX-Gga gene
A map of the Mn-LOX-Gga gene is shown in Fig. 1A, and
important features are summarized in Table 2. About
3.4 kb of the genome of Gga was sequenced,  0.8 kb of
the 5¢-untranslated region (5¢-UTR) (up to the vector
sequence) and  0.6-kb of the 3¢-UTR. The GC content
averaged 60.5%. The 5¢-UTR did not contain TATA or
CAAT-like boxes. The transcription start point for the
Mn-LOX-Gga and Mn-LOX-Ggt genes were determined
by 5¢-RACE (Table 2) and found to be located 72 nucleo-
tides from the tentative translation start point. About 80%
of fungal genes have a purine (usually A) at position )3
from the translation start point [36]. The Mn-LOX-Gga

Glx
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
d
Tyr
Val
65 (64)
34 (33)
61 (59)
3
43 (42)
57 (55)
14
17
58 (56)
24 (23)
6
31 (30)
42 (41)
46 (47)
48 (47)

18
7
33
45 (43)
40 (39)
36 (35)
ND
23 (22)
38 (37)
74 (70)
40 (37)
64
1
45
53
15 (14)
20 (18)
67 (66)
22
10 (9)
33 (32)
38
35 (34)
35
8
23
35 (33)
a
Normalized to 618 and to 602 amino acids, as the mature proteins
may consist of 602 amino acids due to cleavage of a signal peptide

2058
taaagg
Met
1
ArgSerArgIle……PheLeuSerVal
618
Intron 5¢-Donor Branch signal 3¢-Acceptor
Intron I …AGCg
445
tatgtgc t
562
gctaac ggctatag
577
CGT…
…IleThrSer
124
Arg
125
GlyGlyPhe…
a
The transcription start point of Mn-LOX-Ggt gene was a(1)gtaggttc…, and the translation start was …acgaaA(73)TGCGC.
Ó FEBS 2002 Cloning of manganese lipoxygenase (Eur. J. Biochem. 269) 2693
Northern and Southern blot analyses
The cDNA probe hybridized to two poorly separated bands
of  2.7 and  3.1 kb, respectively, of total RNA from Ggt
(Fig. 1B). The polyadenylation sites were not determined,
but the sequenced 0.6 kb of the 3¢-UTR of Mn-LOX-Gga
contained three tentative eukaryotic polyadenylation signals
[37], i.e., A(2174)AUUAA, A(2438)AUAAC, and
C(2577)AUAAA. Southern blot analysis yielded the expec-

When the predicted amino-acid sequence of Mn-LOX-Gga
was subject to
BLAST
search [33], the program reported
homology with the consensus sequence of lipoxygenases
with three-dimensional structure (Pfam 00305, LOX;
and the family
of lipoxygenases. Mammalian LOX yielded the highest
scores, followed by the plant lipoxygenases. A partial
alignment of Mn-LOX with the consensus Pfam LOX is
shown in Fig. 3. The
BLAST
algorithm (with Blosom62)
aligned Mn-LOX with 372 residues of mouse 8S-LOX with
26% identical and 42% similar amino-acid residues. The
corresponding figures for mouse 12S-LOX of leukocyte-
type was 27% and 40% (out of 434 residues). The first 125
amino acids of Mn-LOX showed little homology to LOX;
only the coral 8S-LOX indicated homology of this region.
The coral LOX could be aligned with more than 500
amino acids (residues 83–587) of Mn-LOX with 24%
identical and 39% positive residues. LOX2 of Arabidopsis
thaliana andotherplantLOXcouldalsobealignedwith
about 25–27% amino-acid identity over 300–400 amino
acids, and so could the probable LOX of Pseudomonas
aeruginosa [39].
The homology of Mn-LOX and the LOX gene family
included the two a helices of the latter that contain the
four Fe(II) ligands. These ligands are two His residues
found in ahelix 9 (and in the characteristic sequence of

3
-N(482)-X-G (seventh line of Fig. 3), suggesting
that His478 and Asn482 may have the same function as
these residues have in Fe-LOX. Finally, there appeared
to be conserved amino acids at the C-terminal end of
Mn-LOX (data not shown), but the characteristic
C-terminal isoleucine residue of LOX was not conserved.
The C-terminal amino acid of Mn-LOX was valine, and
there is precedence for the C-terminal valine as a metal
ligand in both native and recombinant Fe-LOX (see
below).
Fig. 3. Partial alignment of Mn-LOX-Gga
precursor with Pfam LOX. The
BLAST
algo-
rithm was used for alignment. The two
sequences were aligned from regions corres-
ponding to the beginning of a helix 6 to the
end of a helix 21 of soybean LOX L1. Red
letters mark identity, blue letters similarity,
and letters in italics mark low complexity. The
Fe ligands of Pfam LOX shown in this align-
ment are His341, His346, His533 and Asn537,
which were aligned with His294, His297,
His478 and Asn482 in the Mn-LOX sequence.
2694 L. Ho
¨
rnsten et al. (Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
We have cloned and sequenced the gene of Mn-LOX of

a helices 9 and 18 of soybean LOX L1 suggested that
Mn(II) could be coordinated to four amino acids in the
same way as Fe(II) in LOX (cf. Fig. 3). The four Mn
ligands were tentatively identified as His290 and His294 of
a helix 9 and His478 and Asn482 of a helix 18. In LOX L1,
this asparagine residue is located with its amide oxygen at
3.05–3.3 A
˚
from Fe, in the various structures, and thus is
considered to be only a weak ligand. By further analogy,
the fifth Mn ligand could be the carboxyl oxygen of the
C-terminal amino acid valine. Rat 5-LOX has valine as the
C-terminal amino acid [42], and site-directed mutagenesis
of murine platelet and leukocyte 12-LOX has shown that
the C-terminal isoleucine may be substituted by valine with
retention of enzyme activity, whereas most other substitu-
tions yielded inactive enzymes [43,44]. A water molecule is
the sixth Fe ligand of soybean LOX-L1 [12], and
Fe
3+
OH
N
has recently been identified as the catalytic base
for hydrogen abstraction [45]. This mechanism is also
plausible for Mn-LOX, as electron paramagnetic resonance
spectra of the X- and W-bands (9.2 and 94 GHz,
respectively) show that the coordination environment of
Mn-LOX is similar to that in Fe-LOX with three N-ligands
to the metal centre and O-ligands in the remainder of the
six coordination positions [46]. These data are consistent

properties. The corresponding studies with insertion of
one or two amino acids into the a helix 9 of Mn-LOX
may also be warranted, but they will only provide
circumstantial evidence until the three-dimensional struc-
ture of Mn-LOX is solved. Studies on expression of
Mn-LOX for this purpose are now in progress.
LOX can cause oxidative degradation of cell membranes,
and plant LOX are often activated by pathogen attack as a
means of pathogen resistance. G. graminis illustrates that an
invading pathogen may secrete Mn-LOX as a means of
pathogenicity. We report that the unique biochemical
properties of Mn-LOX might be related to an unpreceden-
ted structural difference in a conserved region near the metal
center of Fe-LOX rather than to the metal ligands.
ACKNOWLEDGEMENTS
Supported by the Swedish Research Council in Medicine (03X-06523)
and Magn. Bergvalls Stiftelse. We thank A
˚
. Engstro
¨
m, Uppsala
University, for valuable suggestions.
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