RPE65 is a key metalloenzyme responsible for maintaining visual function in vertebrates. Despite extensive research on this membrane-bound retinoid isomerase, fundamental questions regarding its enzymology remain unanswered. Here, we report the crystal structure of RPE65 in a membrane-like environment. These crystals, obtained from enzymatically active, nondelipidated protein, displayed an unusual packing arrangement wherein RPE65 is embedded in a lipid-detergent sheet. Structural differences between delipidated and nondelipidated RPE65 uncovered key residues involved in substrate uptake and processing. Complementary iron K-edge X-ray absorption spectroscopy data established that RPE65 as isolated contained a divalent iron center and demonstrated the presence of a tightly bound ligand consistent with a coordinated carboxylate group. These results support the hypothesis that the Lewis acidity of iron could be used to promote ester dissociation and generation of a carbocation intermediate required for retinoid isomerization.
Figure left: Conformational differences in the membrane-binding surface and active-site entrance observed between lipid-embedded and delipidated RPE65. Arrows pointing from the delipidated structure (gray) to the lipid-embedded structure (yellow) show the general direction of movement between the two structures. Residues F196, F264, and W268 in the lipid-embedded structure form a continuous aromatic surface near the active-site entrance, indicated by the broad green arrow.
Figure right: (E) Experimental EXAFS spectra (dotted line) and best-fit curve (solid line) for detergent-solubilized RPE65.
Kiser PD, Farquhar ER, Shi W, Sui X, Chance MR, Palczewski K.Structure of RPE65 isomerase in a lipidic matrix reveals roles for phospholipids and iron in catalysis. Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):E2747-56. doi: 10.1073/pnas.1212025109. Epub 2012 Sep 24.