Sandbox Reserved 895: Difference between revisions

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==== '''[1.2.2.1] Overall Structural Analysis of RPE65''' ====

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>

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, generated in PyMol and 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_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. Figure generated using PyMol. <ref> DOI 19805034 </ref>,<ref> The PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC. </ref>]]

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

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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. 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. The linear portion of the molecule then occupies the main tunnel. 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 molecules. However, the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).

[[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>]]

[[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. Figure generated using PyMol. <ref> DOI 19805034 </ref>, <ref> The PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC. </ref>]]

=== ''' [1.2.3] Proposed Enzymatic Mechanism of RPE65''' ===

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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.

Comparing bovine RPE65 across different species using a BLAST search showed that RPE65 is highly conserved between animals. <ref> Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/</ref> 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. Figure generated using BLAST<ref> Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/</ref>]]

[[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]]

[[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 <ref> Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/</ref>]]

=== '''[1.3.2] Mutations leading to disease''' ===

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@@ Line 42: / Line 42: @@
 ==== '''[1.2.2.1] Overall Structural Analysis of RPE65''' ====
-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>
+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, generated in PyMol and 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_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. Figure generated using PyMol. <ref> DOI 19805034 </ref>,<ref> The PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC. </ref>]]
 ==== '''[1.2.2.2] Active Site Structural Analysis of RPE65''' ====
@@ Line 54: / Line 54: @@
 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. 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. The linear portion of the molecule then occupies the main tunnel. 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 molecules. However, the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4).
-[[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>]]
+[[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. Figure generated using PyMol. <ref> DOI 19805034 </ref>, <ref> The PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC. </ref>]]
 === ''' [1.2.3] Proposed Enzymatic Mechanism of RPE65''' ===
@@ Line 67: / Line 67: @@
 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.
+Comparing bovine RPE65 across different species using a BLAST search showed that RPE65 is highly conserved between animals. <ref> Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/</ref> 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: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. Figure generated using BLAST<ref> Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/</ref>]]
-[[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]]
+[[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 <ref> Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/</ref>]]
 === '''[1.3.2] Mutations leading to disease''' ===