Plant Seed Peroxygenase Is an Original Heme-oxygenase with an EF-hand Calcium Binding Motif

Abstract

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.

The Reductase Activity of the Arabidopsis Caleosin RESPONSIVE TO DESSICATION20 Mediates Gibberellin-Dependent Flowering Time, Abscisic Acid Sensitivity, and Tolerance to Oxidative Stress

“…However, the identity of RD20 as a peroxygenase is not definitively established. It does possess in its amino acid sequence essential features that typify such enzymes, such as an EF-hand calcium-binding motif and two strictly conserved His residues (Hanano et al, 2006). In addition, RD20 can form FAOHs in conjunction with a-DOX in vitro (Shimada et al, 2014), and a peroxygenase-like activity was detected in crude extracts of salt-stressed Arabidopsis, which was postulated to result from the induction of RD20 by salinity (Partridge and Murphy, 2009).…”

“…RD20 has been suggested to be a peroxygenase (Hanano et al, 2006;Shimada et al, 2014), and thus the enzymatic features of the recombinant protein expressed in yeast (Saccharomyces cerevisiae) were studied. Peroxygenases are typically able to catalyze cooxidations of diverse substrates in the presence of various hydroperoxides (Blée, 1998).…”

“…However, the molecular mechanisms by which distinct caleosins exert their functions remained unknown. The recent identification of ATS1 and CLO4 as peroxygenases (Hanano et al, 2006;Blée et al, 2012) has prompted the suggestion that caleosins might fulfill their physiological functions via their enzymatic production of oxylipins (Partridge and Murphy, 2009;Aubert et al, 2010;Kim et al, 2011). Among the primary oxylipins formed in vitro by peroxygenases are the unsaturated fatty acid hydroxides (FAOHs; Fig.…”

Pyridine as novel substrate for regioselective oxygenation with aromatic peroxygenase from Agrocybe aegerita

“…The term peroxygenase is also used for special plant heme proteins which neither belong to the P450s nor to the peroxidases but show sequence similarity with calcium-binding proteins [33]. Unlike fungal aromatic peroxygenases, plant peroxygenases are integral membrane proteins which are strictly dependent on organic hydroperoxides and preferably oxygenate unsaturated fatty acids [33,34].…”

“…Unlike fungal aromatic peroxygenases, plant peroxygenases are integral membrane proteins which are strictly dependent on organic hydroperoxides and preferably oxygenate unsaturated fatty acids [33,34]. In this context, it is interesting to mention that an unknown peroxide-consuming enzyme from plant tissue cultures was proposed to be responsible for PY hydroxylation at the meta-position [35].…”