Sandbox Reserved 895: Difference between revisions

[[Image:RPE65_Emixustat_and_Palmitate_PyMOL.jpg|thumb|center|512 px|alt=Figure 1B: Emixustat and palmitate ester in Binding Pocket| '''Figure 1B:''' Bovine RPE65 Binding Pocket with Emixustat and palmitate ester generated in PyMOL using PDB: ''4RSV'']]

[[Image:Binding_surface_overlap.jpg|thumb|center|512 px|alt=Figure 1C: Emixustat RPE65 binding pocket opening| '''Figure 1C:''' Bovine RPE65 Opening into Binding Pocket with the electron mesh of Emixustat outlined using PyMOL, PDB: ''4RSV'']]

Following dissociation from opsin, all-''trans''-retinal is transported from the lumen of the photoreceptor disk though an ATP-cassette transporter 4 (''ABCA4''). The enzyme all-''trans''-retinol dehydrogenase (atRDH) reduces all-''trans''-retinal into all-''trans''-retinol. Another transporter enzyme, interphotoreceptor retinoid-binding protein (IRBP) facilitates the transport of all-''trans''-retinol back into the hRPE. Within the hRPE, all-''trans''-retinol is esterified by the enzyme lecithin retinol acyltransferase (LRAT) to form all-''trans''-retinyl ester. RPE65 also known as retinoid isomerohydrolase (IMH) then converts all-''trans''-retinyl ester into 11-''cis''-retinol and palmityl acid. Following the RPE65 catalyzed reaction, 11-''cis''-retinol dehydrogenase (11-''cis''-RDH) oxidizes 11-''cis''-retinol into 11-''cis''-retinal. 11-''cis''-retinal is finally transported back into the photoreceptors to be conjugated to opsin to form the rhodopsin complex. This process is shown in Figure 2. <ref>DOI 10.1016/j.bbadis.2018.04.014</ref>

Crystal structure of bovine RPE65 (PDB: ''3FSN''), which is 99% similar to human RPE65, (although the crystal structure for human RPE65 is not currently available) is used as the basis of studying RPE65 structure. RPE65 resembles a seven-bladed β-propeller with single-stranded extension on blades VI and VII and two-stranded extension on blade III shown in '''Figure 4'''. The top face of the β-propeller is defined by connecting the outer strand of the β-sheet with the inner strand of the next β-sheet. The iron cofactor is located near the top surface of the propeller which is coordinated by four His residues and three secondary Glu residues. Each blade of the propeller contributes one His residue to coordinate with the iron cofactor. A hydrophobic tunnel leads the protein exterior to the active site which is defined by the iron ion to accommodate the passage of retinoids (which are conjugated to a fatty acid tail) from the membrane to the RPE65 catalytic site. The mouth of the tunnel is surrounded by three groups of nonpolar residues that contribute to the overall hydrophobicity of the tunnel and the integration with the lipid bilayer. There are also a few aromatic amino acid side chains that reside in this portion of the enzyme. This suggest that the depth of RPE65 membrane is restricted to the proximal portions of the phospholipid acyl chains with respect to the polar head groups. Arg and Lys residues within this region also contribute to the association with the negatively charged phospholipid head groups. <ref> DOI 19805034 </ref>

[[Image:RPE65_Figure4_S7_blades.jpg|thumb|center|512 px|alt=Figure 4: RPE65 structure| '''Figure 4:''' Crystal structure of bovine RPE65 viewed from the bottom face of the seven-bladed β-propeller labeled in roman numerals from I to VII <ref> DOI 19805034 </ref>]]

[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val. The bond length interactions are shown in angstroms from the iron atom. <ref> DOI 19805034 </ref>]]

From an evolutionary standpoint it might be expected that the RPE65 catalytic mechanism would resemble that of the carotenoid oxygenase as sequence homology places RPE65 in the same family as the CCOs. This is not the case! Although the current enzymatic mechanism for RPE65 is only hypothesized with current research, there is not enough experimental evidence to definitively rule out that RPE65 catalyzes its function with molecular oxygen in the isomerization reaction. However, if such were the case the reaction would require chemistry that is even more complex which is not justifiable by the current scientific evidence. A study by Oberhauser and colleagues in 2008 on NinaB, a member of the CCO family from moths with isomerooxygenase activity was described, indicating that the members of this family originally developed isomerase activity while keeping the carotenoid oxygenase activity. The results of this study suggested a functional link between vertebrate RPE65 and insect NinaB which are both essential for the synthesis of key visual cycle chromophores. The function of NinaB showed that RPE65, a retinoid isomerase in the CCO family, retained the emergent activity that was found in a common ancestor. Comparing the topologies between the enzyme and substrate between NinaB and RPE65 showed that the iron cofactor was not directly involved in the double bond isomerization activity. Although further research is required to determine the ancestry of RPE65, it is nonetheless fascinating to study the evolution and the diversification of a protein family in animals by following the trace from a single multifunctional ancestral protein to several highly specialized enzymes seen in vertebrates. <ref> DOI 19020100 </ref> Using Cluster Omega, the amino acid sequence of NinaB and bovine RPE65 was compared. Although the structure alignment is quite different, there are still some sequence homology between the two enzymes that may suggest an ancestor linkage.  

Comparing bovine RPE65 across different species using a BLAST search showed that RPE65 is highly conserved between animals. From mammals, to rodents there was at least a 97% similarity in sequences. An example shown below in '''Figure 7A''' is a sequence alignment between bovine RPE65 and hamster RPE65. Throughout the sequence alignment there are only 12 amino acid residues that are different between the two species. By showing RPE65 is conserved, researchers are able to use bovine RPE65 to predict the structure and activity of human RPE65 in drug testing and drug development. '''Figure 7B''' shows the lineage of all know RPE65 enzymes.

[[Image:sequence_alignment.jpg|thumb|center|512 px|alt=Figure 7A: bovine and hamster RPE65 sequence alignment| '''Figure 7A:''' Sequence alignment between bovine RPE65 and hamster RPE65 showing highly conserved amino acid residue between organisms.]]

[[Image:Lineage_RPE65_Full.jpg|thumb|center|512 px|alt=Figure 7B: Full RPE65 lineage| '''Figure 7B:''' RPE65 lineage tree using BLAST. Note that the red arrow is a continuation of the lineage tree after "multiple organisms" and is not to scale. Note that the scale changes from 0.01 to 0.006 between the two portions of the lineage]]

[[Media:RPE65_with_emixustat.mp4|Figure 9: 3-dimensional structure of (''R'')-emixustat bound in RPE65 with bond lengths ]]