Philip D. Kiser

Chemistry of the Retinoid (Visual) Cycle

As succinctly summarized by Wolf,1 lack of vitamin A (all-trans-retinol) was recognized by ancient Egyptians as causing a visual deficiency involving the retina and cornea that could be cured by eating liver. One of the symptoms of vitamin A deficiency is night blindness or nyctalopia (from Greek νύκτ-, nykt – night; and αλαός, alaos – blindness), recognized by ancient Greeks, including Hippocrates, as affecting the retina.2 In 1913 McCollum showed that “fat-soluble factor A” was essential for growth of a rat colony (reviewed in ref (3)). The treatment of factor A-deficiency included liver or liver extracts, but later in 1930 Moore found that yellow pigment (carotene) was a good substitute for this therapy.4 A major breakthrough occurred in 1931 when the chemical structures for β,β-carotene and retinol (its all-trans isomer now known as vitamin A) were determined by Karrer and colleagues.5 But, it was Wald who discovered that retinol derivatives constitute the chemical basis of our vision,6 a contribution subsequently recognized by a Nobel Prize award in 1967. In 1950–1960, a variety of vitamin A metabolic transformations, including oxidation/reduction and esterification, were elucidated by Olson,7−20 Goodman,21−32 Chytil and Ong,33−41 and Norum and Blomhoff.42−57 The discovery that one set of these metabolites, namely retinoic acids, plays a key role in the nuclear regulation of a large number of genes added a notable dimension to our knowledge of gene expression. This mechanism is also a critical player in the successful healing of corneal wounds,58 a second manifestation of vitamin A-deficiency recognized earlier. Further progress in understanding the multiple physiological roles of retinoids has been made in recent years, due mainly to the successful application of modern scientific technology. Examples include enzymology combined with structural biology, in vivo imaging based on retinoid fluorescence, improvements in analytical methods, generation and testing of animal models of human diseases with specific pathogenic genetics, genetic analysis of human conditions related to changes in vitamin A metabolism, and pharmacological approaches to combat these diseases. In this review we focus on the involvement of retinoids in supporting vision via light-sensitive rod and cone photoreceptor cells in the retina. We begin with a brief description of isopentenyl diphosphate (IPP) biosynthesis, which is essential for carotenoid (C40 isoprenoid) production. Certain of these colored compounds, such as lutein, are deposited in our retina’s macula, appearing as a “yellow” spot. Other carotenoids containing at least one unmodified β-ionone ring (represented by β,β-carotene and cryptoxanthin) serve as precursors of all-trans-retinal. Many different compounds can be generated from this monocyclic diterpenoid, which contains a β-ionone ring and polyene chain with a C15 aldehyde group. Among the numerous enzymatic activities that contribute to retinoid metabolism, polyene trans/cis isomerization is a particularly fascinating reaction that occurs in specialized structures of the retina based on a two cell system comprised of retinal photoreceptor cells and the retinal pigment epithelium (RPE). A specific enzyme system, called the retinoid (visual) cycle, has evolved to accomplish retinoid isomerization that is required for visual function in vertebrates. Individual enzymes of this pathway harbor secrets about the molecular mechanisms of this chemical transformation. Malfunctions of these processes or other pathological reactions often precipitate severe retinal pathologies. This review attempts to balance contributions that have been published over the past decades and does not intend to replace the views of investigators with different perspectives of retinoid chemistry in the eye.59−85

Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-Å resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.

Key enzymes of the retinoid (visual) cycle in vertebrate retina.

A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.

Metabolic Basis of Visual Cycle Inhibition by Retinoid and Nonretinoid Compounds in the Vertebrate Retina

In vertebrate retinal photoreceptors, the absorption of light by rhodopsin leads to photoisomerization of 11-cis-retinal to its all-trans isomer. To sustain vision, a metabolic system evolved that recycles all-trans-retinal back to 11-cis-retinal. The importance of this visual (retinoid) cycle is underscored by the fact that mutations in genes encoding visual cycle components induce a wide spectrum of diseases characterized by abnormal levels of specific retinoid cycle intermediates. In addition, intense illumination can produce retinoid cycle by-products that are toxic to the retina. Thus, inhibition of the retinoid cycle has therapeutic potential in physiological and pathological states. Four classes of inhibitors that include retinoid and nonretinoid compounds have been identified. We investigated the modes of action of these inhibitors by using purified visual cycle components and in vivo systems. We report that retinylamine was the most potent and specific inhibitor of the retinoid cycle among the tested compounds and that it targets the retinoid isomerase, RPE65. Hydrophobic primary amines like farnesylamine also showed inhibitory potency but a short duration of action, probably due to rapid metabolism. These compounds also are reactive nucleophiles with potentially high cellular toxicity. We also evaluated the role of a specific protein-mediated mechanism on retinoid cycle inhibitor uptake by the eye. Our results show that retinylamine is transported to and taken up by the eye by retinol-binding protein-independent and retinoic acid-responsive gene product 6-independent mechanisms. Finally, we provide evidence for a crucial role of lecithin: retinol acyltransferase activity in mediating tissue specific absorption and long lasting therapeutic effects of retinoid-based visual cycle inhibitors.

Structural basis of carotenoid cleavage: from bacteria to mammals.

Carotenoids and their metabolic derivatives serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, photoprotection and photosynthesis as well as cell signaling. These organic compounds are also important for visual function in vertebrate and non-vertebrate organisms. Enzymatic transformations of carotenoids to various apocarotenoid products are catalyzed by a family of evolutionarily conserved, non-heme iron-containing enzymes named carotenoid cleavage oxygenases (CCOs). Studies have revealed that CCOs are critically involved in carotenoid homeostasis and essential for the health of organisms including humans. These enzymes typically display a high degree of regio- and stereo-selectivity, acting on specific positions of the polyene backbone located in their substrates. By oxidatively cleaving and/or isomerizing specific double bonds, CCOs generate a variety of apocarotenoid isomer products. Recent structural studies have helped illuminate the mechanisms by which CCOs mobilize their lipophilic substrates from biological membranes to perform their characteristic double bond cleavage and/or isomerization reactions. In this review, we aim to integrate structural and biochemical information about CCOs to provide insights into their catalytic mechanisms.

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

Membrane-binding and enzymatic properties of RPE65.

Regeneration of visual pigments is essential for sustained visual function. Although the requirement for non-photochemical regeneration of the visual chromophore, 11-cis-retinal, was recognized early on, it was only recently that the trans to cis retinoid isomerase activity required for this process was assigned to a specific protein, a microsomal membrane enzyme called RPE65. In this review, we outline progress that has been made in the functional characterization of RPE65. We then discuss general concepts related to protein-membrane interactions and the mechanism of the retinoid isomerization reaction and describe some of the important biochemical and structural features of RPE65 with respect to its membrane-binding and enzymatic properties.

Structural basis for the acyltransferase activity of lecithin:retinol acyltransferase−like proteins

Background: The enzymology of aminophospholipid-processing enzymes is not well understood. Results: Structures of HRAS-like tumor suppressors resemble those of thiol proteases with Cys-His-His catalytic triad. Conclusion: An aminophospholipid acyl group first modifies the Cys residue of HRAS-like tumor suppressor enzymes before it is transferred onto a second substrate. Significance: This study provides a structural basis for the enzymatic mechanism of HRAS-like tumor suppressors. Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.

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

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.

Stereospecificity of Retinol Saturase: Absolute Configuration, Synthesis, and Biological Evaluation of Dihydroretinoids

Retinol saturase carries out a stereospecific saturation of the C13−C14 double bond of all-trans-retinol to generate (13R)-all-trans-13,14-dihydroretinol. This compound is found in cells expressing mouse or zebrafish retinol saturase and in the livers of mice fed retinyl palmitate. All-trans-13,14-dihydroretinol is oxidized in vivo to all-trans-13,14-dihydroretinoic acid, a highly selective agonist of the retinoic acid receptor. The naturally occurring (13R)-all-trans-13,14-dihydroretinoic acid is a weaker agonist than the (13S) enantiomer, indicating enantioselective recognition by the ligand-binding pocket of this receptor. Consequently the (13S) enantiomer, acting through the retinoic acid receptor, also inhibits adipose differentiation more potently than the (13R) enantiomer.