Sandbox Reserved 895: Difference between revisions

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From an enzymatic point of view, RPE65 is similar to its carotenoid-cleaving relatives in that a ferrous ion is required for catalytic activity. Although the specific enzymatic mechanism for RPE65 is complex and not well elucidated, previous research has shown that the most probable reaction mechanism is as shown in '''Figure 6A''' and '''Figure 6B'''. The initial interaction with the ester moiety of all-trans-retinyl ester with the ferrous ion polarizes the carbon-15 oxygen σ (sigma) bond and makes the fatty ester an activated leaving group. Formation of a resonance-stabilized carbocation reduces the π (pi) bond order of the system allowing temporary rotation about the carbon-11, carbon-12 σ bond. A water molecule obtained from the bulk solution attacks the carbon-15 atom quenching the carbocation intermediate. Finally, the protonation of the fatty acid carboxylate group promotes its dissociation from the ion cofactor. This process releases the fatty acid and the now 11-cis-retinol from the active site of RPE65. <ref> DOI 19805034 </ref>

== '''[1.3] Evolutionary Conservation''' ==

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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 even more complex chemistry 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 would be needed to determine the ancestry of RPE65, it is fascinating to study the evolution and the diversification of 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>

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

Mutations within the RPE65 enzyme can impact the enzyme catalyzed reactions that is pertinent to the retinoid cycle. Disease such as Leber congenital amaurosis (LCA) or retinal dystrophy disease such as retinitis pigmentosa (RP) arise due to the mutations in RPE65. Often these diseases can lead to impaired vision or even blindness. Since human and bovine RPE65 share approximately 99% of the amino acid sequences, it can be assumed that the secondary and tertiary structures are similar. This relationship allows us to map human LCA and RP to the bovine RPE65 amino acid sequences. In LCA and RP patients, it was noted that most of the amino acid mutations were adjacent to the blades of the β-propeller rather than in the connecting loops and helices. Blade VII was the most common area of mutation. Since mutations here would alter how the β-propeller seals into its proper structure, this could impact the location and residues that contribute to chelating the iron ion. For patients with LCA, missense mutations caused a major change in the first and second shell of the iron ion ligands which is essential for RPE65 isomerase activity. As such patients with these mutations had non-functional RPE65 enzymes. Amino acid Arg91, Tyr368 and His182 were most often mutated in patients with RPE-65 associated LCA or RP. This showed that these amino acid positions were integral to the function of the enzymatic activity. Mutations such as His182Arg or Tyr368His caused major structural deformations that altered the accommodation of the substituted side chain. In such cases, enzyme activity was decreased or lost. Arg91 is known to be important in positioning RPE65 to the membrane-binding elements. Arg91 also forms a salt bridge with Glu127 which is located on the C-terminal side of the enzyme. This interaction is critical for subcellular localization. As a result, mutations here alteres the ability of RPE65 for subcellular localization. <ref> DOI 19805034 </ref>

= '''[2] Protein-Ligand Interaction''' =

RPE65 interacts with other ligands to catalyze an enzymatic isomerhydrolase activity.

== '''[2.1] Endogenous Ligand, all-trans-retinyl ester''' ==

The endogenous ligand for human RPE65 is all-''trans''-retinyl ester. Binding to the active site as well as the catalytic mechanism were mentioned above in the sections ''[1.2.2.2] Active Site Structural Analysis of RPE65'' and ''[1.2.3] Proposed Enzymatic Mechanism of RPE65'' respectively.

== '''[2.2] Exogenous Ligand, emixustat''' ==

=== ''' [2.2.1] (R)-emixustat (ACU-4429), Competitive Antagonist''' ===

==== '''[2.2.1.1] Development''' ====

Emixustat (ACU-4429) shown in '''Figure 7''' is an investigational small molecule inhibitor of RPE65 first invented by a British-American chemist, Ian L. Scott. When synthesized, emixustat usually presents of a racemic mixture of (''R'')-emixustat and (''S'')-emixustat. The (''R'')-isomer is associated with increased binding affinity and potency and is used in drug development and research. Formulated as a hydrochloride salt, (''R'')-emixustat hydrochloride is taken by mouth and functions as a visual cycle modulator (VCM) in atrophic (dry) age related macular degeneration (AMD). (''R'')-emixustat has been shown to reduce toxic retinal byproducts in the retinoid cycle such as N-retinylidiene-N-retinylethanolamine (A2E).

= References =

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@@ Line 56: / Line 56: @@
 From an enzymatic point of view, RPE65 is similar to its carotenoid-cleaving relatives in that a ferrous ion is required for catalytic activity. Although the specific enzymatic mechanism for RPE65 is complex and not well elucidated, previous research has shown that the most probable reaction mechanism is as shown in '''Figure 6A''' and '''Figure 6B'''. The initial interaction with the ester moiety of all-trans-retinyl ester with the ferrous ion polarizes the carbon-15 oxygen σ (sigma) bond and makes the fatty ester an activated leaving group. Formation of a resonance-stabilized carbocation reduces the π (pi) bond order of the system allowing temporary rotation about the carbon-11, carbon-12 σ bond. A water molecule obtained from the bulk solution attacks the carbon-15 atom quenching the carbocation intermediate. Finally, the protonation of the fatty acid carboxylate group promotes its dissociation from the ion cofactor. This process releases the fatty acid and the now 11-cis-retinol from the active site of RPE65. <ref> DOI 19805034 </ref>
-[[Image:RPE65_Figure6_Reacation_mechanism.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Hypothesized Reaction Mechanism| '''Figure 6:''' (A) All-''trans''-retinol (B) Hypothesized RPE65 Reaction Mechanism <ref> DOI 19805034 </ref>]]
+[[Image:RPE65_Figure6_Reacation_mechanism.jpg|thumb|center|512 px|alt=Figure 6: RPE65 Hypothesized Reaction Mechanism| '''Figure 6:''' (A) All-''trans''-retinol (B) Hypothesized RPE65 Reaction Mechanism <ref> DOI 19805034 </ref>]]
 == '''[1.3] Evolutionary Conservation''' ==
@@ Line 62: / Line 62: @@
 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 even more complex chemistry 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 would be needed to determine the ancestry of RPE65, it is fascinating to study the evolution and the diversification of 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>
+=== '''[1.3.2] Mutations leading to disease''' ===
+Mutations within the RPE65 enzyme can impact the enzyme catalyzed reactions that is pertinent to the retinoid cycle. Disease such as Leber congenital amaurosis (LCA) or retinal dystrophy disease such as retinitis pigmentosa (RP) arise due to the mutations in RPE65. Often these diseases can lead to impaired vision or even blindness. Since human and bovine RPE65 share approximately 99% of the amino acid sequences, it can be assumed that the secondary and tertiary structures are similar. This relationship allows us to map human LCA and RP to the bovine RPE65 amino acid sequences. In LCA and RP patients, it was noted that most of the amino acid mutations were adjacent to the blades of the β-propeller rather than in the connecting loops and helices. Blade VII was the most common area of mutation. Since mutations here would alter how the β-propeller seals into its proper structure, this could impact the location and residues that contribute to chelating the iron ion. For patients with LCA, missense mutations caused a major change in the first and second shell of the iron ion ligands which is essential for RPE65 isomerase activity. As such patients with these mutations had non-functional RPE65 enzymes. Amino acid Arg91, Tyr368 and His182 were most often mutated in patients with RPE-65 associated LCA or RP. This showed that these amino acid positions were integral to the function of the enzymatic activity. Mutations such as His182Arg or Tyr368His caused major structural deformations that altered the accommodation of the substituted side chain. In such cases, enzyme activity was decreased or lost. Arg91 is known to be important in positioning RPE65 to the membrane-binding elements. Arg91 also forms a salt bridge with Glu127 which is located on the C-terminal side of the enzyme. This interaction is critical for subcellular localization. As a result, mutations here alteres the ability of RPE65 for subcellular localization. <ref> DOI 19805034 </ref>
+= '''[2] Protein-Ligand Interaction''' =
+RPE65 interacts with other ligands to catalyze an enzymatic isomerhydrolase activity.
+== '''[2.1] Endogenous Ligand, all-trans-retinyl ester''' ==
+The endogenous ligand for human RPE65 is all-''trans''-retinyl ester. Binding to the active site as well as the catalytic mechanism were mentioned above in the sections ''[1.2.2.2] Active Site Structural Analysis of RPE65'' and ''[1.2.3] Proposed Enzymatic Mechanism of RPE65'' respectively.
+== '''[2.2] Exogenous Ligand, emixustat''' ==
+=== ''' [2.2.1] (R)-emixustat (ACU-4429), Competitive Antagonist''' ===
+==== '''[2.2.1.1] Development''' ====
+Emixustat (ACU-4429) shown in '''Figure 7''' is an investigational small molecule inhibitor of RPE65 first invented by a British-American chemist, Ian L. Scott. When synthesized, emixustat usually presents of a racemic mixture of (''R'')-emixustat and (''S'')-emixustat. The (''R'')-isomer is associated with increased binding affinity and potency and is used in drug development and research. Formulated as a hydrochloride salt, (''R'')-emixustat hydrochloride is taken by mouth and functions as a visual cycle modulator (VCM) in atrophic (dry) age related macular degeneration (AMD). (''R'')-emixustat has been shown to reduce toxic retinal byproducts in the retinoid cycle such as N-retinylidiene-N-retinylethanolamine (A2E).
+[[Image:Figrue7_Emixustat.jpg|thumb|center|512 px|alt=Figure 7: Emixustat| '''Figure 7:''' (''R'')-Emixustat]]
 = References =
 <references/>