RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle.
The isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle and is essential for normal vision. Recently, we have established that protein RPE65 is the isomerohydrolase catalyzing this reaction. The present study investigated if metal ions are required for the isomerohydrolase activity of RPE65. The conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol was used as the measure for isomerohydrolase activity. Metal chelators 2,2'-bipyridine and 1,10-phenanthroline both showed dose-dependent inhibitions of the isomerohydrolase activity in bovine RPE microsomes, with IC50 values of 0.5 and 0.2 mm, respectively. In the same reaction systems, however, lecithin-retinol acyltransferase (LRAT) activity was not affected by these metal chelators. The isomerohydrolase activity inhibited by the metal chelators was restored by FeSO4 but not by CuSO4, ZnCl2, or MgCl2. Moreover, addition of Fe(III) citrate or FeCl3 did not restore the activity, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity. To confirm this result in recombinant RPE65, we expressed RPE65 in a 293A cell line stably expressing LRAT. In vitro activity assay showed that both metal chelators inhibited isomerohydrolase activity of recombinant RPE65. The addition of FeSO4 restored the enzymatic activity of the recombinant RPE65. Further, two specific iron-staining methods showed that purified RPE65 contains endogenous iron. Inductively coupled plasma mass spectrometry measurements showed that bovine RPE65 binds iron ion with a stoichiometry of 0.8 +/- 0.1. These results indicate that RPE65 is an iron-dependent isomerohydrolase in the visual cycle.
Moiseyev
Gennadiy
G
Department of Cell Biology, Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Takahashi
Yusuke
Y
Chen
Ying
Y
Gentleman
S
S
Redmond
T Michael
TM
Crouch
Rosalie K
RK
Ma
Jian-Xing
JX
D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D005136:Eye Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005296:Ferrous Compounds / Q000494:pharmacology*; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007536:Isomerism; D008861:Microsomes; D012176:Retinoids / Q000378:metabolism*; D014795:Visual Pathways; D014801:Vitamin A / Q000378:metabolism
D002614:Chelating Agents; D005136:Eye Proteins; D005296:Ferrous Compounds; D012176:Retinoids; D014801:Vitamin A; D006867:Hydrolases