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

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

Cloning of the manganese lipoxygenase gene reveals homology

with the lipoxygenase gene family

Lena Ho¨rnsten1, Chao Su1, Anne E Osbourn2, Ulf Hellman3and Ernst H Oliw1

1

Department of Pharmaceutical Biosciences, Uppsala Biomedical Centre, Uppsala, Sweden;2The Sainsbury Laboratory,

John Innes Centre, Norwich, UK;3Ludwig 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 graminisyielded 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-X15

-H-X4-H-X3-E of a helix 9, one histidine and usually an

asparaine residue in the sequence H-X3-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-X14-H(294)-X3-H(297)-X3-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)-X3-N(482)-X-G] could be aligned with the two metal ligands of a helix 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: Ernst.Oliw@farmbio.uu.se

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)

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

E X P E R I M E N T A L P R O C E D U R E S

Materials

[a-32P]dCTP (3000 CiÆmmol)1), dNTPs, [a-33P]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 mM 3-[cyclohexylamine]-1-propane sulfonic acid (pH 11) with 10% methanol (v/v) (100 V, 4 h at 21C) 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 (55C 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 lM each primer, 10 mM Tris/HCl pH 8.3, 50 mM KCl, 3.0 mM MgCl2, 0.2 mM dNTPs and 1.5 U TaqDNA polymerase The PCR protocol was: 94C for 3 min, 1 cycle, followed by

94C for 45 s, 48 C for 45 s, 72 C for 1 min for 30 cycles, a final extension step (72C, 10 min) and then cooling to 8C 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

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 with32P using

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 SpeI and NsiI 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/0.22M formaldehyde gels, transferred to

Hybond-N membranes and hybridized in QuikHyb

(Strat-agene) with the 32P-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 NotI and HindIII

Homology search

The gapped BLAST algorithm of the GenBank at NCBI

(http://www.ncbi.nlm.nih.gov; [33]) was used for database

search and for pair wise alignments, whereas theLASERGENE

MEGALIGN program (Dnastar, Madison, WI, USA) was

used for multiple alignments

R E S U L T S

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¢- and 3¢-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, which were expected to yield a 1.4 kb fragment The latter was detected as shown Size markers are shown by arrows.

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).cAnalysed 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 pointa Translation start pointa 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…

GlyGlyPhe…

a

The transcription start point of Mn-LOX-Ggt gene was a(1)gtaggttc…, and the translation start was …acgaaA(73)TGCGC.

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

618 amino acids and had a molecular mass of 67.7 kDa

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

and Mn-LOX-Gga differed only at seven amino acids, 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 search [33], the program reported

homology with the consensus sequence of lipoxygenases with three-dimensional structure (Pfam 00305, LOX; http://www.ncbi.nlm.nih.gov/Structure/cdd) 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 thalianaand other plant LOX could also be aligned with 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 a helix 9 (and in the characteristic sequence of

30 amino acids: WLLAK-X15-H-X4-H-X3-E), one His residue and a distant ligand (usually an asparagine residue) in a helix 18 (and in the characteristic sequence WI-X4-H-X3-N-X-GQ) [11–15,40] The region of Mn-LOX that correspondeds to a helix 9 of Mn-LOX, contained the sequence WLLAK-X14-H(290)-X3-H(294)-X3-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-X4 -H(478)-X3-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.

D I S C U S S I O N

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

precursor is smaller than mammalian LOX ( 73 kDa)

and much smaller than plant LOX ( 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 b barrel 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

Fe3+OHNhas 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 two Fe(II) ligands can be aligned with all known LOX 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

A C K N O W L E D G E M E N T S

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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