Structure of RPE65 isomerase in a lipidic matrix reveals roles for phospholipids and iron in catalysis

Abstract

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 Xray 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.metalloprotein | monotopic membrane protein | extended X-ray absorption fine structure | isomerohydrolase V ision is a complex, multistep process whereby environmental information contained in light rays is converted to chemical and electrical signals in the retina that are relayed to the brain for interpretation. The initial step in this process is mediated by light receptors called rod and cone opsins and their associated G proteins in photoreceptor cells (1). These receptors contain a vitamin A-derived chromophore called 11-cis-retinal that undergoes cis to trans isomerization upon absorption of a photon. Isomerization of the chromophore converts the receptor to a state in which it can activate downstream signaling molecules that alter the electrochemical state of the photoreceptor cell (2, 3). Following photoisomerization, the Schiff base-linked chromophore is hydrolyzed and dissociates from the receptor. Continuous regeneration of 11-cis-retinal, required for maintenance of visual function, is critically dependent on a metabolic pathway known as the visual cycle (4, 5). At the heart of this cycle is the enzymatic conversion of all-transretinyl esters to 11-cis-retinol by the retinoid isomerase, RPE65, mutations of which cause the severe childhood blinding disease Leber congenital amaurosis (6, 7).RPE65 is an iron-dependent, membrane-bound enzyme expressed nearly exclusively in the retinal pigment epithelium (RPE) (8, 9). The iron dependence of RPE65 was anticipated on the basis of its evolutionarily relationship to carotenoid cleavage oxygenases (CCOs). However, CCO activity has not been demonstrated for RPE65, and it is likely that iron serves a different, albeit poorly understood, function in this enzyme. It is now appreciated that phospholipids constitute a second critical factor necessary for RPE65 activity. Purified RPE65 exhibited retinoid isomerase activity only when bound to all-trans-retinyl ester-incorporated membrane vesicles (10). Moreover, destruction of intact microsomal phosp...

Expansion of First-in-Class Drug Candidates That Sequester Toxic All-Trans-Retinal and Prevent Light-Induced Retinal Degeneration

“…Interestingly, introduction of an extra nitrogen atom (QEA-G-001-NH 2 and QEA-G-002-NH 2 ) moderately recovered the inhibitory properties. This observation supports the previous hypothesis that the isomerization occurs via a carbocation intermediate, and that the positively charged compound inhibits the reaction (Golczak et al, 2005b;Kiser et al, 2009Kiser et al, , 2012Kiser et al, , 2014.…”

“…Although LRAT was shown to have a broad substrate specificity (Canada et al, 1990), chemical boundaries that determine the substrate selectivity for this enzyme had not been clarified. In contrast, the crystal structure of RPE65 was elucidated in detail (Kiser et al, 2009(Kiser et al, , 2012, revealing a narrow tunnel that leads into the active site of this enzyme. Indeed, a relatively small structural modification of the retinoid moiety could effectively abolish binding of an inhibitor to this enzyme.…”

Utilization of Dioxygen by Carotenoid Cleavage Oxygenases

“…Even though O 2 does not stoichiometrically participate in this proposed reaction, a major reduction in O 2 concentration, as accomplished by our deoxygenation procedure, should still greatly slow the rate of retinoid isomerization, which is not what we observed. The lack of activity reduction after deoxygenation also cannot be explained by potential tight binding of O 2 to the iron center as our prior structural and spectroscopic studies of RPE65 did not reveal such a stable Fe-O 2 complex (20,44). Most importantly, a recent structural determination of RPE65 in complex with a retinoid mimetic indicates that the polyene chain of carotenoids binds at a position distant from the iron center, which excludes formation of an Fe-O 2 -retinoid complex (26).…”

Analysis of Carotenoid Isomerase Activity in a Prototypical Carotenoid Cleavage Enzyme, Apocarotenoid Oxygenase (ACO)

“…The typical yield was 3-6 mg of purified protein per liter of E. coli culture (Table 1). Our previous x-ray absorption spectroscopy studies on this asisolated sample suggested that the ACO catalytic iron was in the ferrous state (32). To confirm that the as-isolated sample obtained by our protocol was enzymatically active, we measured its steadystate kinetic parameters.…”