Functional Characterization of a CruP-Like Isomerase in Dunaliella.

Dunaliella bardawil is a marine unicellular green algal that produces large amounts of ß-carotene and is a model organism for studying the carotenoid synthesis pathway. However, there are still many mysteries about the enzymes of the D. bardawil lycopene synthesis pathway that have not been revealed. Here, we have identified a CruP-like lycopene isomerase, named DbLyISO, and successfully cloned its gene from D. bardawil. DbLyISO showed a high homology with CruPs. We constructed a 3D model of DbLyISO and performed molecular docking with lycopene, as well as molecular dynamics testing, to identify the functional characteristics of DbLyISO. Functional activity of DbLyISO was also performed by overexpressing gene in both E. coli and D. bardawil. Results revealed that DbLyISO acted at the C-5 and C-13 positions of lycopene, catalyzing its cis-trans isomerization to produce a more stable trans structure. These results provide new ideas for the development of a carotenoid series from engineered bacteria, algae, and plants.

Retinal pigment epithelium 65 kDa protein (RPE65): An update.

Vertebrate vision critically depends on an 11-cis-retinoid renewal system known as the visual cycle. At the heart of this metabolic pathway is an enzyme known as retinal pigment epithelium 65 kDa protein (RPE65), which catalyzes an unusual, possibly biochemically unique, reaction consisting of a coupled all-trans-retinyl ester hydrolysis and alkene geometric isomerization to produce 11-cis-retinol. Early work on this isomerohydrolase demonstrated its membership to the carotenoid cleavage dioxygenase superfamily and its essentiality for 11-cis-retinal production in the vertebrate retina. Three independent studies published in 2005 established RPE65 as the actual isomerohydrolase instead of a retinoid-binding protein as previously believed. Since the last devoted review of RPE65 enzymology appeared in this journal, major advances have been made in a number of areas including our understanding of the mechanistic details of RPE65 isomerohydrolase activity, its phylogenetic origins, the relationship of its membrane binding affinity to its catalytic activity, its role in visual chromophore production for rods and cones, its modulation by macromolecules and small molecules, and the involvement of RPE65 mutations in the development of retinal diseases. In this article, I will review these areas of progress with the goal of integrating results from the varied experimental approaches to provide a comprehensive picture of RPE65 biochemistry. Key outstanding questions that may prove to be fruitful future research pursuits will also be highlighted.

RPE65 Palmitoylation: A Tale of Lipid Posttranslational Modification.

RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65's membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.

Structure and biochemical studies of a pseudomonad maleylpyruvate isomerase from Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 can utilize various aromatic hydrocarbons as a carbon source. Among the three genes involved in the gentisate pathway of P. aeruginosa, the gene product of PA2473 belongs to the ζ-class glutathione S-transferase and is predicted to be a maleylpyruvate isomerase. In this study, we determined the crystal structure of maleylpyruvate isomerase from Pseudomonas aeruginosa PAO1 (PaMPI) at a resolution of 1.8 Å. PaMPI functions as a dimer and shows the glutathione S-transferase fold. The structure comparison with other glutathione S-transferase structures enabled us to predict the glutathione cofactor binding site and suggests that PaMPI has differences in residues that make up the putative substrate binding site. Biochemical study of PaMPI showed that the protein has an MPI activity. Interestingly, unlike the reported glutathione S-transferases so far, the purified PaMPI showed isomerase activity without the addition of the reduced glutathione, although the protein showed much higher activity when the glutathione cofactor was added to the reaction mixture. Taken together, our studies reveal that the gene product of PA2473 functions as a maleylpyruvate isomerase and might be involved in the gentisate pathway.

Transcriptional regulation of fatty acid cis-trans isomerization in the solvent-tolerant soil bacterium, Pseudomonas putida F1.

One key to the success of Pseudomonas spp. is their ability to reside in hostile environments. Pseudomonas spp. possess a cis-trans isomerase (Cti) an enzyme that converts the cis-unsaturated fatty acids (FAs) of the membrane lipids to their trans-isomers to rigidify the membrane and thereby resist stresses. Whereas the posttranslational Cti regulation has been previously reported, transcriptional cti regulation remains to be studied in more details. Here, we have studied cti transcriptional regulation in the solvent-tolerant strain Pseudomonas putida F1. Two cti transcriptional start sites (cti-279 and cti-77) were identified with cti-279 transcript being dominant. Expression of cti was found to increase with temperature increase, addition of the organic solvent, octanol and in the stationary growth phase. We found that cti expression was repressed by the cyclic-AMP receptor protein (Crp) and repression required the cyclic-AMP ligand of Crp. Production of trans-unsaturated FAs was found to decrease after 24 h of growth. Although this decrease was accompanied by an increase in cyclopropane FA content, this was not at the expense of trans-unsaturated FAs demonstrating the absence of competition between Cti and Cfa in FA modification.

Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65.

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of these knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.

Mutations in the MIT3 gene encoding a caroteniod isomerase lead to increased tiller number in rice.

Carotenoids not only play important roles in light harvesting and photoprotection against excess light, but also serve as precursors for apocaroteniod hormones such as abscisic acid (ABA) and strigolactones (SLs). Although light- and ABA-associated phenotypes of the carotenoid biosynthesis mutants such as albino, leaf variegation and preharvest sprouting have been studied extensively, the SLs-related branching phenotype is rarely explored. Here we characterized four allelic rice mutants named mit3, which exhibited moderately increased tiller number, semi-dwarfism and leaf variegation. Map-based cloning revealed that MIT3 encodes a carotenoid isomerase (CRTISO), the key enzyme catalyzing the conversion from prolycopene to all-trans-lycopene in carotenoid biosynthesis. Prolycopene was accumulated while all-trans-lycopene was barely detectable in the dark-grown mit3 seedlings. Accordingly, content of lutein and ß-carotene, the two most abundant carotenoids, was significantly reduced. Furthermore, content of epi-5DS, a native SL, was significantly reduced in mit3. Exogenously applied GR24, a synthetic SL, could rescue the tillering phenotype of mit3. Double mutant analysis of mit3 with the SLs biosynthesis mutant d17 revealed that MIT3 controls tiller development upstream of the SLs biosynthesis pathway. Our results reveal that the tillering phenotype of mit3 is due to SL deficiency and directly link carotenoid deficiency with SL-regulated rice tillering.

Rational Tuning of Visual Cycle Modulator Pharmacodynamics.

Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.

In silico Mapping of Protein Unfolding Mutations for Inherited Disease.

The effect of disease-causing missense mutations on protein folding is difficult to evaluate. To understand this relationship, we developed the unfolding mutation screen (UMS) for in silico evaluation of the severity of genetic perturbations at the atomic level of protein structure. The program takes into account the protein-unfolding curve and generates propensities using calculated free energy changes for every possible missense mutation at once. These results are presented in a series of unfolding heat maps and a colored protein 3D structure to show the residues critical to the protein folding and are available for quick reference. UMS was tested with 16 crystal structures to evaluate the unfolding for 1391 mutations from the ProTherm database. Our results showed that the computational accuracy of the unfolding calculations was similar to the accuracy of previously published free energy changes but provided a better scale. Our residue identity control helps to improve protein homology models. The unfolding predictions for proteins involved in age-related macular degeneration, retinitis pigmentosa, and Leber's congenital amaurosis matched well with data from previous studies. These results suggest that UMS could be a useful tool in the analysis of genotype-to-phenotype associations and next-generation sequencing data for inherited diseases.

Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism.

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 µM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.

Biochemical characterization and selective inhibition of ß-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by ß-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 µm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 µm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.

A History of the Classical Visual Cycle.

The visual cycle, the biochemical process by which the light-sensitive isomer of vitamin A is continually recycled, is crucial to vision in a healthy eye. More than 150 years of research into this remarkable biochemical process has given invaluable understanding in debilitating visual diseases that impact thousands of individuals worldwide, many of them children. The visual cycle spans photoreceptor cells in the retina and the underlying retinal pigment epithelium (RPE) and requires a protein called RPE65 for its function. In many ways, RPE65 is the capstone to the cyclical processing of vitamin A in the eye, and the discovery of this retinol isomerase helped fill a critical gap in the understanding of retinoid processing in vision. This chapter will focus on the history of visual cycle research, from the first experiments well over a century ago to the discovery of RPE65. Because of the undeniable importance of RPE65 in the visual cycle, this chapter will also focus on the protein structure and mechanism by which it converts light-insensitive all-trans-vitamin A to light-sensitive 11-cis-vitamin A for continued visual function. Finally, this chapter will briefly discuss RPE65 and its known disease associations in the clinical setting. Thanks to the efforts of researchers for well over a century in studying the visual cycle, the medical community is now poised to make significant gains in the treatment of blindness.

Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

Molecular pharmacodynamics of emixustat in protection against retinal degeneration.

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

Catalytic mechanism of a retinoid isomerase essential for vertebrate vision.

Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

Identification of key residues determining isomerohydrolase activity of human RPE65.

RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met(1)-Arg(33)) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.

Rescue of enzymatic function for disease-associated RPE65 proteins containing various missense mutations in non-active sites.

Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.

Computational investigations on the catalytic mechanism of maleate isomerase: the role of the active site cysteine residues.

The maleate isomerase (MI) catalysed isomerization of maleate to fumarate has been investigated using a wide range of computational modelling techniques, including small model DFT calculations, QM-cluster approach, quantum mechanical/molecular mechanical approach (QM/MM in the ONIOM formalism) and molecular dynamics simulations. Several fundamental questions regarding the mechanism were answered in detail, such as the activation and stabilization of the catalytic Cys in a rather hydrophobic active site. The two previously proposed mechanisms were considered, where either enediolate or succinyl-Cys intermediate forms. Small model calculations as well as an ONIOM-based approach suggest that an enediolate intermediate is too unstable. Furthermore, the formation of succinyl-Cys intermediate via the nucleophilic attack of Cys76(-) on the substrate C2 (as proposed experimentally) was found to be energetically unfeasible in both QM-cluster and ONIOM approaches. Instead, our results show that Cys194, upon activation via the substrate, acts as a nucleophile and Cys76 acts as an acid/base catalyst, forming a succinyl-Cys intermediate in a concerted fashion. Indeed, the calculated PA of Cys76 is always higher than that of Cys194 before or upon substrate binding in the active site. Furthermore, the mechanism proceeds via multiple steps by substrate rotation around C2-C3 with the assistance of the now negatively charged Cys76, leading to the formation of fumarate. Finally, our calculated barrier is in good agreement with experiment. These findings represent a novel mechanism in the racemase superfamily.

Structural approaches to understanding retinal proteins needed for vision.

The past decade has witnessed an impressive expansion of our knowledge of retinal photoreceptor signal transduction and the regulation of the visual cycle required for normal eyesight. Progress in human genetics and next generation sequencing technologies have revealed the complexity behind many inherited retinal diseases. Structural studies have markedly increased our understanding of the visual process. Moreover, technical innovations and improved methodologies in proteomics, macromolecular crystallization and high resolution imaging at different levels set the scene for even greater advances. Pharmacology combined with structural biology of membrane proteins holds great promise for developing innovative accessible therapies for millions robbed of their sight or progressing toward blindness.