Báo cáo Y học: Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family doc

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

over 350–400 amino-acid residues of the catalytic domains.
Lipoxygenases have one water molecule and five amino
acids as Fe ligands. These are two histidine residues in the
highly conserved 30 amino-acid sequence WLLAK-X
15
-
H-X
4
-H-X
3
-E of a helix 9, one histidine and usually an
asparaine residue in the sequence H-X
3
-N-X-G of a helix 18,
and the carboxyl oxygen of the C-terminal isoleucine (or
valine) residue. The homologous sequence of a helix 9 of
Mn-Lipoxygenase [WLLAK-X
14
-H(294)-X
3
-H(297)-X
3
-E]
contained two single-amino-acid gaps, but otherwise His294
and His297 aligned with the two His residues, which
coordinate iron. Mn-Lipoxygenase [H(478)-X
3
-N(482)-X-G]
could be aligned with the two metal ligands of ahelix 18,
and the C-terminal residue was Val618. We conclude that

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

molecule and five amino acids [12]. The iron ligands are
the carboxyl oxygen of the C-terminal isoleucine (or
valine) residue, the nitrogen atoms of two histidine
residues of a helix 9 and one histidine residue of
a helix 18, and the distant amid oxygen of an asparagine
residue of a helix 18 [1,12,16]. There is a large group of
nonheme Fe(II) enzymes, which have a common struc-
tural motif, the 2-His-1-carboxylate facial triad. This triad
designates two histidine nitrogens and the carboxyl
oxygen of asparagine, glutamic acid, or the C-terminal
isoleucine residue as three of the Fe ligands, and LOX is
considered to belong to this group of enzymes, although
LOX has five metal ligands [17].
Early reports suggest that LOX occur in fungi, but the
enzymes have not been described in detail (for a review see
[18]). The take-all fungus, Gaeumannomyces graminis,
which is a root pathogen of wheat, forms the only fungal
LOX that has been characterized. This LOX has several
unique properties [19]. First, it contains Mn in its active
center and it was therefore designated Mn-LOX.
Manganese is tightly bound to the apoenzyme in a 1 : 1
stoichiometry, and cannot be extracted with metal chela-
tors. Second, the enzyme metabolizes linoleic and
a-linolenic acids to 13R-hydroperoxy fatty acids and to
novel LOX products, 11S-hydroperoxy fatty acids [20].
Correspondence to E. H. Oliw, Division of Biochemical
Pharmacology, Department of Pharmaceutical Biosciences, Uppsala
University, PO Box 591, Husargatan 3, SE-751 24 Uppsala, Sweden.
Fax: + 46 18 55 29 36, Tel.: + 46 18 471 44 55,
E-mail:

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-

genase) occurs in two homologous forms with either
prosthetic Fe or Mn [22]. X-ray crystallography of the Fe
form of the 2,3-dioxygenase shows that Fe is coordinated
to three amino-acid residues (His145, His209 and Glu260)
and to two molecules of water [23,24]. Site-directed
mutagenesis of the Mn form suggests that the correspond-
ing conserved amino acids (His155, His214 and Glu266)
are essential for catalytic activity [25]. Superoxide dismu-
tases also have identical metal ligands and tertiary fold for
Fe- and Mn-dependent forms [26]. This left us with the
intriguing possibility that Mn-LOX and LOX might have
identical metal ligands and yet form different oxidation
products.
The aim of the present investigation was to clone and
sequence the Mn-LOX gene.
EXPERIMENTAL PROCEDURES
Materials
[a-
32
P]dCTP (3000 CiÆmmol
)1
), dNTPs, [a-
33
P]ddNTPs,
Hybond-N membranes, DNA labeling beads (dCTP), and
T-primed first-strand kit were from Amersham Pharmacia
Biotech. TA cloning kits were from Invitrogen. Taq DNA
polymerase and the enhanced avian RT/PCR kit were from
Sigma. Restriction enzymes were from New England
BioLabs. Two strains of G. graminis [var avenae (Gga)

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

and blotted onto polyvinyldifluoride membranes. Elec-
trophoretic transfer (Mini Trans-Blot, Bio-Rad) was in
10 m
M
3-[cyclohexylamine]-1-propane sulfonic acid
(pH 11) with 10% methanol (v/v) (100 V, 4 h at 21 °C).
The membranes were stained for proteins with Coomassie
blue [29]. The excised protein band of Mn-LOX was subject
to total amino-acid analysis.
Amino-acid sequencing
Purified Mn-LOX from Ggt was subject to in situ digestion
in the SDS/PAGE gel with Lys-C, trypsin, and with V8
protease [30]. Peptides were isolated by narrow-bore RP-
HPLC on the Smart System (Amersham Pharmacia Bio-
tech) and subject to amino-acid sequencing (PerkinElmer
ABI 494 Sequencer). Analysis of the C-terminal amino-acid
sequence was performed as described previously [31].
RT/PCR analysis and cloning
Mycelia of G. graminis were harvested by filtration. Total
RNA was prepared by grinding of mycelia in liquid
nitrogen, extracting with the RNeasy plant kit, and
checking for integrity by agarose gel electrophoresis. About
2.5 lg of total RNA and 1 U enhanced avian reverse
transcriptase (Sigma) in 20 lL were used for first-strand
synthesis (55 °C for 50 min) according to the manufac-
turer’s protocol, and 4 lL were used as templates for each
PCR. For 5¢-RACE, total RNA (1 lg) was transcribed
with a gene-specific primer (Mns21r, 5¢-CTGGCTGG
GGGGTGTACTTCTTCT-3¢) according to the protocol
from Life Technologies for 5¢-RACE of GC-rich templates.

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

of the genomic library was performed in QuikHyb (Strat-
agene) as described [28,32]. Three rounds of screening
purified positive plaques. The Bluescript plasmids were
rescued from the Bluescript SK phagemid with helper phage
(Stratagene).
Restriction analysis
Analysis of the Bluescript plasmids was performed with
restriction enzymes followed by size-fractioning in 0.8–1.5%
agarose gels. SpeIandNsiI yielded a DNA segment
( 3 kb), which contained the coding region of the
Mn-LOX gene. This segment was subcloned into pGEM-
5Zf(+) (Promega).
Northern and Southern blot analyses
Total RNA (15 lg) was size-fractionated by electrophoresis
in 1% agarose/0.22
M
formaldehyde gels, transferred to
Hybond-N membranes and hybridized in QuikHyb (Strat-
agene) with the
32
P-labeled cDNA probe (337 bp, see
below) as described previously [32]. The DNA fragment,
which was obtained by cleavage of the genomic sequence of
Mn-LOX with BamHI and NotI was used as a probe (see
Fig. 1). Genomic DNA of Gga was isolated and  1.7 lg
was digested with NotIandHindIII.
Homology search
The gapped
BLAST
algorithm of the GenBank at NCBI

(; [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

GGTAGAAGAGGCCCTGRCAVGC-3¢), whereas the
tryptic peptide-2 (HPVMGVLNR) provided the sense
primer EO3a (5¢-CATCCSGTSATGGGYGTSCTBAA-3¢)
and the antisense primer EOr3a (5¢-CGGTTSAGGACRC
CCATVACVGGRTG-3¢). The internal peptide sequences
of the remaining eight peptides (the C-terminal peptide,
GLSQGMPFWTALNPAVNPFFLSV; VDDAFAAPDL
LAGNGPGRA; EMAGRGFDGGLSQG; TNVGADLT
YTPLDD; FSGVLPLHPAWL; QAVEQVSLLAR; GLV
GEDSGPR; LFLVDHSYQK) could be identified in the
deduced protein sequences of Mn-LOX (Fig. 2).
RT-PCR
cDNA was initially prepared from Ggt. The primers Mn60
and EOr3A generated a band of 230 bp, which contained
Fig. 1. Organization of the Mn-LOX-Gga gene, Northern and Southern
blot analyses. (A) The Mn-LOX-Gga gene. The open box indicates the
protein coding region. The solid lines show the 5¢-and3¢-UTR and the
intron. An arrow marks start of transcription and some restriction sites
are marked. A solid line shows the two overlapping cDNA fragments,
which were obtained by RT/PCR and used for screening of the
genomic library. (B) Northern blot analysis of Ggt yielded a major
signal at  2.7 kb and a minor signal at  3.1 kb. Size markers are
from the RNA ladder. (C) Southern blot analysis of Ggt.Genomic
DNA was digested with BamHI and NotI,whichwereexpectedto
yield a 1.4 kb fragment. The latter was detected as shown. Size markers
are shown by arrows.
2692 L. Ho
¨
rnsten et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the deduced sequence WLLAK, which is well conserved in

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

gene had A in this position, whereas the Mn-LOX-Ggt gene
had G (Table 2). cDNA analysis also showed the presence
of an intron of 133 bp. The exon/intron borders followed
the gt/ag rule. There was a typical signal (TGCTAAC;
consensus c/TNCTA/GAC/t) for branching that occurs in
splicing of RNA of filamentous fungi located 25 nucleotides
from the 3¢ acceptor. The intron was short, a characteristic
of filamentous fungi [36].
Table 1. Total amino-acid compositions of Mn-LOX and their precur-
sors.
Amino
acids
Mn-LOX-Ggt
a
Mn-LOX-Gga
a
Measured
618 (602)
Deduced
b
618 (602)
Measured
c
618 (602)
Deduced
618 (602)
Ala
Arg
Asx
Cys

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)

10
21 (20)
37 (36)
74 (70)
40 (37)
65
1
45
53
15 (14)
17 (15)
66 (65)
21
11 (10)
33 (32)
38
35 (34)
36
8
23
37 (35)
67 (66)
26 (25)
59 (57)
ND
56 (55)
63 (61)
8
21
69 (67)

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

(MRSRILAIVFAARHVA) [38].
b
Deduced Mn-LOX precursor
from partial sequencing of cDNA of Ggt and the sequenced
C-terminal peptide (Fig. 2).
c
Analysed after blotting to poly(viny-
lidene difluoride) membranes, which may give artificially low values
for Arg, His, and Lys [48].
Fig. 2 The predicted amino-acid sequence of
the Mn-LOX-Gga precursor. Amino acids are
numbered beginning with the methionine
residue (Met1) of Mn-LOX-Gga. Internal
peptides generated by cleavage of Mn-LOX-
Ggt with endoproteinases are underlined. The
amino-acid sequence of Mn-LOX-Ggt
differed from Mn-LOX-Gga in only seven
positions (K52N, V258A, I384V, I473V,
L493V, A507T, and I586M).
Table 2. Translation, transcription and termination sequences of the Mn-LOX-Gga gene and the exon–intron borders.
Transcription start point
a
Translation start point
a
Translation end
a
1
gcaggttc… acaaaA
73
TGCGC……GAGCGTC

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-

ted signal at  1.4 kb (Fig. 1C), which was in agreement
with a single Mn-LOX gene, but this was not investigated
further.
Deduced amino-acid sequences
The predicted amino-acid sequence of the Mn-LOX-Gga
precursor based on an open reading frame of 1854
nucleotides is shown in Fig. 2, and it contained the 10
sequenced peptides as shown. The precursor thus contained
618aminoacidsandhadamolecularmassof67.7kDa.
The gene was isolated from a library of Gga, whereas
peptide information was obtained from Mn-LOX-Ggt. We
also partially sequenced cDNA of Ggt from the 5¢-end
(598 amino acids). In combination with the sequenced
C-terminal peptide of 23 amino acids of Mn-LOX-Ggt
(Fig. 2), we obtained the complete amino-acid sequence.
The two proteins were almost identical, as Mn-LOX-Ggt
andMn-LOX-Ggadifferedonlyatsevenaminoacids,two
of which were found in the sequenced peptides: Ile384 [in
GLV(384)GEDSGPR] and Ile586 [in EM(586)AGRGFD
GGLSQG].
The N-terminal sequence of Mn-LOX sequences was
recently reported in a patent [38], which showed that an
N-terminal peptide was released by cleavage between Ala16
and Ala17. This tentative signal peptide, MRS-
RILAIVFAARHVA, thus contains four alanine and three
arginine residues, which might explain the low number of
alanine and arginine residues in the two native Mn-LOX
compared to the number of these residues of their deduced
precursors (Table 1).
Sequence homology

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

30 amino acids: WLLAK-X
15
-H-X
4
-H-X
3
-E), one His
residue and a distant ligand (usually an asparagine
residue) in ahelix 18 (and in the characteristic sequence
WI-X
4
-H-X
3
-N-X-GQ) [11–15,40]. The region of Mn-
LOX that correspondeds to a helix 9 of LOX, contained
the sequence WLLAK-X
14
-H(290)-X
3
-H(294)-X
3
-E (3rd
line in Fig. 3). Although this sequence contained only 28
amino acids, the two critical histidine residues appeared
to be conserved, suggesting that His290 and His294 are
Mn(II) ligands. The region of Mn-LOX, which corres-
ponded to a helix 18, contained the sequence WI-X
4
-
H(478)-X

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

G. graminis and the corresponding cDNA. Our main
finding is that Mn-LOX belongs to the LOX gene family
with a unique difference in one of the conserved regions.
The deduced protein sequence of the Mn-LOX precursor
contained 618 amino acids (67.7 kDa). The Mn-LOX
precursorissmallerthanmammalianLOX( 73 kDa)
andmuchsmallerthanplantLOX( 90 kDa). The
central part of the deduced protein sequence of Mn-LOX
(over 450 amino acids) could be aligned with  27–28%
amino-acid identity and  40% similarity with mammalian
LOX (e.g. 15-LOX type 2, 8 and 12-LOX), and with the
three-dimensional consensus LOX sequence (Pfam LOX;
Fig. 3).
Plant and mammalian LOX contain a small N-terminal
b-barrel domain and a large C-terminal and catalytic
domain, as revealed by three-dimensional analysis [11,15].
The function of the b barrel is unknown, but it is identical in
connectivity to the C-terminal domain of certain lipases and
might be related to lipid binding [15] and to membrane
translocation [41]. The b barrels of plant LOX consist of
 150 amino acids and the b barrel of mammalian LOX
contains  125 amino acids. In spite of the conserved three-
dimensional structure, the plant and mammalian amino-
acid sequences of the bbarrel domains cannot be aligned
with significant homology [15]. It was therefore not
unexpected that the N-terminal 85 amino acids of Mn-LOX
failed to align with plant and mammalian LOX. Three-
dimensional analysis is needed to determine whether
Mn-LOX also contains an N-terminal b barrel.
Alignment of the C-terminal domain of Mn-LOX along

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

with nitrogen atoms of the three histidine residues and
oxygen atoms of valine, asparagine, and water. X-ray
crystallography will be needed to conclusively confirm that
His294, His297, His478, Asn482, Val618 and water are
ligands of Mn(II), but this seems likely from the well
established sequence homology of LOX, the electron
paramagnetic resonance spectra, and the precedence of
conserved metal ligands in Fe and Mn forms of other
homologous enzymes [23–26].
The major part of a helix 9 of soybean LOX L1 with its
twoFe(II)ligandscanbealignedwithallknownLOX
without amino-acid gaps [13,35,47]. Alignment of a helix 9
with Mn-LOX yielded two single-amino-acid gaps, one
between the characteristic motif WLLAK and His290,
and the other between His290 and His294 (Fig. 3). As
regards a helix 18 and the C-terminal amino acid, there
appeared to be no principle differences between Mn-LOX
and other LOX. The unprecedented sequence difference in
a helix 9 between all published LOX sequences and
Mn-LOX is therefore probably of paramount importance
for the geometry at the metal center and the metal
specificity. It may explain the paradox that Fe- and
Mn-LOX can have conserved metal ligands, yet form
different oxidation products and abstract hydrogen in
different ways [19,20]. It will clearly be of interest to
combine molecular modeling with site-directed mutagen-
esis of LOX with known three-dimensional structure (e.g.
soybean LOX-L1, LOX-L3 or rabbit 15-LOX) to deter-
mine the impact of deleting one or two amino acids in
a helix 9 on the metal center and on the catalytic

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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