Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase.
Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.
Psylinakis
E
E
Department of Chemistry, University of Crete, Heraklion, Crete, Greece.
Davoras
E M
EM
Ioannidis
N
N
Trikeriotis
M
M
Petrouleas
V
V
Ghanotakis
D F
DF
D000446:Aldehyde-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D010945:Plants, Edible / Q000201:enzymology* / Q000235:genetics; D011190:Potassium Cyanide / Q000737:chemistry; D013053:Spectrophotometry
D005290:Ferric Compounds; D005296:Ferrous Compounds; D007093:Imidazoles; D009569:Nitric Oxide; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase; D011190:Potassium Cyanide