Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family.
Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal 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-Lipoxygenase 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 alpha helix 9, one histidine and usually an asparaine residue in the sequence H-X3-N-X-G of alpha helix 18, and the carboxyl oxygen of the C-terminal isoleucine (or valine) residue. The homologous sequence of alpha 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 alpha 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 alpha helix 9 of lipoxygenases rather than to the metal ligands.
Hörnsten
Lena
L
Department of Pharmaceutical Biosciences, Uppsala Biomedical Centre, Uppsala, Sweden.
Su
Chao
C
Osbourn
Anne E
AE
Hellman
Ulf
U
Oliw
Ernst H
EH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics*; D005658:Fungi / Q000235:genetics*; D008024:Ligands; D008084:Lipoxygenase / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D005656:Fungal Proteins; D008024:Ligands; C112608:manganese lipoxygenase; D008084:Lipoxygenase