Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif.
A growing body of evidence indicates that phytooxylipins play important roles in plant defense responses. However, many enzymes involved in the biosynthesis of these metabolites are still elusive. We have purified one of these enzymes, the peroxygenase (PXG), from oat microsomes and lipid droplets. It is an integral membrane protein requiring detergent for its solubilization. Proteinase K digestion showed that PXG is probably deeply buried in lipid droplets or microsomes with only about 2 kDa at the C-terminal region accessible to proteolytic digestion. Sequencing of the N terminus of the purified protein showed that PXG had no sequence similarity with either a peroxidase or a cytochrome P450 but, rather, with caleosins, i.e. calcium-binding proteins. In agreement with this finding, we demonstrated that recombinant thale cress and rice caleosins, expressed in yeast, catalyze hydroperoxide-dependent mono-oxygenation reactions that are characteristic of PXG. Calcium was also found to be crucial for peroxygenase activity, whereas phosphorylation of the protein had no impact on catalysis. Site-directed mutagenesis studies revealed that PXG catalytic activity is dependent on two highly conserved histidines, the 9 GHz EPR spectrum being consistent with a high spin pentacoordinated ferric heme.
Hanano
Abdulsamie
A
Laboratoire des Phytooxlipines, Institut de Biologie Moléculaire des Plantes-CNRS-UPR 2357, 67083 Strasbourg Cedex, France.
Burcklen
Michel
M
Flenet
Martine
M
Ivancich
Anabella
A
Louwagie
Mathilde
M
Garin
Jérôme
J
Blée
Elizabeth
E
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D018554:Avena / Q000201:enzymology / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D020832:EF Hand Motifs; D004578:Electron Spin Resonance Spectroscopy; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D008055:Lipids / Q000302:isolation & purification; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment
D002135:Calcium-Binding Proteins; D008055:Lipids; D010940:Plant Proteins; C402836:caleosin protein, Sesamum indicum; D006899:Mixed Function Oxygenases; C022333:peroxygenase; D006419:Heme Oxygenase (Decyclizing); D010100:Oxygen; D002118:Calcium