Sandbox Reserved 895: Difference between revisions

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In order to stimulate the phototransduction pathway, 11-''cis''-retinal must be absorbed into the body. 11-''cis''-retinal often referred as Vitamin A, is an important dietary vitamin that starts the cascade of phototransduction. Once absorbed into the body and brought to the photoreceptor outer segment (POS), 11-''cis''-retinal is conjugated to opsin to form the rhodopsin complex. Upon light incidence, 11-''cis''-retinal is converted though a photo-isomerization reaction into all-''trans''-retinal. The conversion of 11-''cis''-retinal to all-''trans''-retinal causes a conformation change in the rhodopsin complex. This conformation change activates a G-protein coupled protein transducing which triggers the subsequent phototransduction cascade. The generated all-''trans''-retinal then dissociates from the rhodopsin complex which frees the opsin protein to bind another 11-''ci''s-retinal molecule and restart the phototransduction cascade. All-''trans''-retinal must be converted back into 11-''cis''-retinal to serve as the chromophore for the phototransduction cascade. To regenerate 11-''cis''-retinal a series of enzyme catalyzed chemical reactions must take place. These reactions take place within the photoreceptor outer segment (hPOS) as well as the retinal pigment epithelium (hRPE).

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 transporter 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>

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According to SCOP, the bovine RPE65 (PDB: ''4RSC'', SCOP: ''8051041''), belongs in the domain 8051041, the family 4007172 corresponding to retinoid isomerase RPE65-like, the superfamily 3002594 corresponding to RPE65-like, the fold 2001013 corresponding to 7-bladed beta-propeller and the class 100001 corresponding to all beta proteins. <ref> SCOP Databank 14 Apr 2020 Available from: http://scop.mrc-lmb.cam.ac.uk/term/8051041 </ref>

Using sequence homology, RPE65 belong to a family of proteins known as carotenoid cleavage oxygenase (CCO) enzymes. This class of enzymes often cleave β-carotene or apocarotenoids. However, what makes RPE65 unique form all the other enzymes in this family is that RPE65 catalyzes an isomerhydrolase reaction. Additionally, unlike the other enzymes in the CCO family, there is no obvious role for molecular oxygen in the RPE65 enzymology. All members of the CCO family contain four conserved histidine residues (His180, His241, His313 and His527) that bind to an ion (Fe2+) cofactor. <ref> DOI 19805034 </ref> <ref> DOI 10.1016/j.bbadis.2018.04.014 </ref>

RPE65 can exist as both soluble and membrane bound forms which can undergo post-translational modifications (PTMs). At cystine residues Cys231, Cys239 and Cys330 the enzyme can be S-palmitoylated. S-palmitoylation of RPE65 was speculated to increase the ratio of membrane-bound to soluble RPE65 which can increase the enzymatic activity. <ref> DOI 15186777 </ref> However other studies have challenged this hypothesis and as such palmitoylation of RPE65 require further research to determine the activity. <ref> DOI 17504753 </ref>

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==== '''[1.2.2.2] Active Site Structural Analysis of RPE65''' ====

Like stated before the RPE65 iron cofactor is found near the top surface of the β-propeller structure. The iron ion is directly coordinated by the Nitrogen-ε atoms of His180, His241, His313 and His527 with an average bond length of 2.2 angstroms. The geometry of the iron ion coordination is that of an octahedral with two open coordination sties. With the exception of His180, the other coordination interactions (His241, His313 and His527) is via hydrogen bonding from the Nδ-H bond on histidine to the Oε on Glu148, Glu417 and Glu469 respectively. The Nδ-H bond on His180 coordinates with a water molecule that is found lower axial, in the hydrophilic region of the propeller. These hydrogen bonds stabilize the octahedral coordination of the iron ion to facilitate the catalysis reaction. Of the two open coordination sties, one of the sites is occupied by the Cγ atom from Val134 located approximately 4.9 angstroms away. These interactions are shown in '''Figure 5A'''.

As stated, the iron ion is also accessible through a main tunnel that enters the helical cap of the protein and is almost orthogonal relative to the propeller axis show in '''Figure 4C'''. This tunnel passes though the metal ion forming a bent cavity within the protein. A secondary tunnel within RPE65 leads from the exterior portion of the protein to the active site but contains a narrow segment that occludes the passage of retinoid substrates as well as other substrates catalyzed by RPE65. Although this second tunnel occludes larger compounds, water, small molecules and ions are still permitted into the active site via this tunnel. As such it is hypothesized that the reactants and the products enter and exit from the same tunnel. The presence of amino acid residues such as Phe, Tyr and Trp within the main tunnel help confer enzyme rigidity as well as stabilize the intermediates of the isomerhydrolase reaction. The hydrophobicity of the cavity once again promotes the participation of lipophilic retinoids from the membrane into the active site as well as encourage the reaction of RPE65 with the membrane.

The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. Shown in '''Figure 5B''', the first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry while the linear portion occupies the main tunnel. Also shown in '''Figure 5B''', the second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecule. However the electron dese region cannot accommodate an longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).

= References =

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@@ Line 15: / Line 15: @@
 In order to stimulate the phototransduction pathway, 11-''cis''-retinal must be absorbed into the body. 11-''cis''-retinal often referred as Vitamin A, is an important dietary vitamin that starts the cascade of phototransduction. Once absorbed into the body and brought to the photoreceptor outer segment (POS), 11-''cis''-retinal is conjugated to opsin to form the rhodopsin complex. Upon light incidence, 11-''cis''-retinal is converted though a photo-isomerization reaction into all-''trans''-retinal. The conversion of 11-''cis''-retinal to all-''trans''-retinal causes a conformation change in the rhodopsin complex. This conformation change activates a G-protein coupled protein transducing which triggers the subsequent phototransduction cascade. The generated all-''trans''-retinal then dissociates from the rhodopsin complex which frees the opsin protein to bind another 11-''ci''s-retinal molecule and restart the phototransduction cascade. All-''trans''-retinal must be converted back into 11-''cis''-retinal to serve as the chromophore for the phototransduction cascade. To regenerate 11-''cis''-retinal a series of enzyme catalyzed chemical reactions must take place. These reactions take place within the photoreceptor outer segment (hPOS) as well as the retinal pigment epithelium (hRPE).
 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 transporter 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>
@@ Line 30: / Line 29: @@
 According to SCOP, the bovine RPE65 (PDB: ''4RSC'', SCOP: ''8051041''), belongs in the domain 8051041, the family 4007172 corresponding to retinoid isomerase RPE65-like, the superfamily 3002594 corresponding to RPE65-like, the fold 2001013 corresponding to 7-bladed beta-propeller and the class 100001 corresponding to all beta proteins. <ref> SCOP Databank 14 Apr 2020 Available from: http://scop.mrc-lmb.cam.ac.uk/term/8051041 </ref>
 Using sequence homology, RPE65 belong to a family of proteins known as carotenoid cleavage oxygenase (CCO) enzymes. This class of enzymes often cleave β-carotene or apocarotenoids. However, what makes RPE65 unique form all the other enzymes in this family is that RPE65 catalyzes an isomerhydrolase reaction. Additionally, unlike the other enzymes in the CCO family, there is no obvious role for molecular oxygen in the RPE65 enzymology. All members of the CCO family contain four conserved histidine residues (His180, His241, His313 and His527) that bind to an ion (Fe2+) cofactor. <ref> DOI 19805034 </ref> <ref> DOI 10.1016/j.bbadis.2018.04.014 </ref>
 RPE65 can exist as both soluble and membrane bound forms which can undergo post-translational modifications (PTMs). At cystine residues Cys231, Cys239 and Cys330 the enzyme can be S-palmitoylated. S-palmitoylation of RPE65 was speculated to increase the ratio of membrane-bound to soluble RPE65 which can increase the enzymatic activity. <ref> DOI 15186777  </ref> However other studies have challenged this hypothesis and as such palmitoylation of RPE65 require further research to determine the activity. <ref> DOI 17504753 </ref>
@@ Line 44: / Line 41: @@
 [[Image:RPE65_Figure4_Structure.jpg|thumb|center|512 px|alt=Figure 4: RPE65 structure| '''Figure 4:''' Structure of RPE65 <ref> DOI 19805034 </ref>]]
+==== '''[1.2.2.2] Active Site Structural Analysis of RPE65''' ====
+Like stated before the RPE65 iron cofactor is found near the top surface of the β-propeller structure. The iron ion is directly coordinated by the Nitrogen-ε atoms of His180, His241, His313 and His527 with an average bond length of 2.2 angstroms. The geometry of the iron ion coordination is that of an octahedral with two open coordination sties. With the exception of His180, the other coordination interactions (His241, His313 and His527) is via hydrogen bonding from the Nδ-H bond on histidine to the Oε on Glu148, Glu417 and Glu469 respectively. The Nδ-H bond on His180 coordinates with a water molecule that is found lower axial, in the hydrophilic region of the propeller. These hydrogen bonds stabilize the octahedral coordination of the iron ion to facilitate the catalysis reaction. Of the two open coordination sties, one of the sites is occupied by the Cγ atom from Val134 located approximately 4.9 angstroms away. These interactions are shown in '''Figure 5A'''.
+As stated, the iron ion is also accessible through a main tunnel that enters the helical cap of the protein and is almost orthogonal relative to the propeller axis show in '''Figure 4C'''. This tunnel passes though the metal ion forming a bent cavity within the protein. A secondary tunnel within RPE65 leads from the exterior portion of the protein to the active site but contains a narrow segment that occludes the passage of retinoid substrates as well as other substrates catalyzed by RPE65. Although this second tunnel occludes larger compounds, water, small molecules and ions are still permitted into the active site via this tunnel. As such it is hypothesized that the reactants and the products enter and exit from the same tunnel. The presence of amino acid residues such as Phe, Tyr and Trp within the main tunnel help confer enzyme rigidity as well as stabilize the intermediates of the isomerhydrolase reaction. The hydrophobicity of the cavity once again promotes the participation of lipophilic retinoids from the membrane into the active site as well as encourage the reaction of RPE65 with the membrane.
+The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. Shown in '''Figure 5B''', the first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry while the linear portion occupies the main tunnel. Also shown in '''Figure 5B''', the second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecule. However the electron dese region cannot accommodate an longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).
+[[Image:RPE65_Figure5_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 Binding Pocket <ref> DOI 19805034 </ref>]]
 = References =
 <references/>