Now You See It- A Retinoid Isomerase Catalytic Mechanism

(a) Investigation of structure and (b, c) inhibitory effects of emixustat and MB-001

Nikki Bilgram

ChemBio Spotlight #3

Paper: Catalytic Mechanism of a Retinoid Isomerase Essential for Vertebrate Vision

http://www.nature.com/nchembio/journal/v11/n6/full/nchembio.1799.html

RPE65 is a membrane-bound retinoid isomerase that is important in the retinoid cycle pathway for vision in vertebrates. It converts fatty acid all-trans-retinyl ester into 11-cis-retinol for rod and cone opsins, which are receptors that receive light and turn it into an electrical signal used for vision. However, a catalytic mechanism for RPE65 has yet to be determined since it is unclear how retinoids bind to its active site. Previous research has led to the discovery of RPE65 active site residues, however possible suggestions for how RPE65 and retinoid interact place retinoid far from these residues in the active site pocket. Within the past year, Kiser et. al have proposed a catalytic mechanism for RPE65 in the retinoid cycle pathway.

Since it has been difficult to obtain a crystal structure of an RPE65 complex (RPE65 + substrate) due to the poor aqueous solubility of retinoids, the authors began their study by finding a molecule that is similar to a retinoid, but also has better solubility. The chemical structure of emixustat was studied, and it was determined that is was a good retinoid mimic along with another molecule, MB-001, that is a hybrid of emixustat.

The authors then tested the whether emixustat and MB-001 bind to RPE65 and inhibit production of 11-cis-retinol using an activity assay, which the authors found that they do. This led to an investigation of RPE65’s active site, binding sites, and hydrophobic interactions with these molecules, which allowed the authors to propose a catalytic mechanism for RPE65, which included acyl and substrate binding, van der Waals, hydrophobic, and electrostatic interactions, ester group polarization, and carbocation stabilization. This showed that derivatives of substrates that have increased solubility, therefore interacting better with active site polar pockets, can be used in other structural studies to determine catalytic mechanisms.

(a) Investigation of structure and (b, c) inhibitory effects of emixustat and MB-001
(a) Investigation of structure and (b, c) inhibitory effects of emixustat and MB-001

11 Replies to “Now You See It- A Retinoid Isomerase Catalytic Mechanism”

  1. Very interesting article! You mentioned that emixustat was a good mimic of RPE65, and I am curious how similar their structures actually are? Are there any differences that might cause mechanisms to differ between the two such as exchanges between polar/nonpolar or basic/acidic amino acids? Additionally, were there other tests mentioned to increase solubility of RPE65 to an extent that allowed for any type of crystal structure to be found?

    1. Glad you liked it!

      It actually wasn’t that emixustat was a good mimic for RPE65 itself, it was a good mimic for a retinoid that would bind to RPE65, forming an RPE65 complex. In regards to similarity of structures, emixustat matched up well to the carbon backbone of RPE65; it has a positively charged amine nitrogen that lines up with the C15 atom of RPE65 which the authors figured would mimic the the C15 cation transition states of RPE65, making it a good molecule with which RPE65 could form a complex.

      What caused a major difference was the fact that emixustat had one less methylene than the product RPE65’s step in the retinoid cycle pathway. This prevented steric and electrostatic clashes with another product of the retinoid cycle pathway, palmitate. By allowing for an interaction between the emixustat and palmitate, the way in which a retinyl ester (which the emixustat is mimicking) binds to RPE65 could be determined.

      There weren’t any other tests to increase the solubility of molecules binding to RPE65.

  2. This is a really cool crystal paper. It is amazing how much information we can derive from a crystal structure: building on previous data these authors provide an entire catalytic mechanism! I was wondering why it is so important to replenish 11-cis-retinal as part of the vision process? Are there any disorders characterized by a deficiency in 11-cis-retinal or RPE65?

    1. Isn’t a great paper!? I was really impressed with the authors!

      11-cis-retinal is essential for rod and cone opsins, which are light-sensitive proteins that bring about the conversion of a photon of light into an electrochemical signal, and this is the first step in the visual transduction cascade. 11-cis-retinal itself, however, forms half of rhodopsin, which is a visual pigment. All of this is important for visual perception; it is needed to interpret visual stimuli detected so we can see.

      In regards to disorders, it doesn’t seem as though it’s the deficiency in 11-cis-retinal that is the primary problem, it’s the mutation in RPE65. A loss-of-function mutation in RPE65 is the cause of the childhood disorder Leber Congenital Amaurosis, which causes blindness due to loss of cone photoreceptors.

  3. Really interesting paper! I was curious as to what emixustant exactly was and why they chose to use it in this test and found that is actually being used as a treatment for AMD due to its inhibitory properties of RPE65. However, it would seem that this would actually cause increased vision loss due to the decreased 11-cis-retinol production. Is there a alternate pathway that can be utilized to allow for the regeneration of the rods/cones necessary for vision so this does not occur?

    1. I’m glad you found it interesting!

      Emixustant is a molecule that mimics a retinoid in that it binds to RPE65. They chose to use it in this test because it’s aqueous solubility properties are greater than those of retinoid itself. This allowed them to more easily make a crystal structure of an RPE65 complex in order to see the binding site activity RPE65, so they could find a catalytic mechanism for which RPE65 works in the retinoid cycle pathway. I hope that clarifies the role of emixustat in this experiment!

      As far as emixustat being used as a treatment for AMD, it inhibiting the loss-of-function mutation that occurs in RPE65 in this disease, and it only partially blocks the activity of RPE65. That was all the information that was given in the article regarding therapeutics, it was mostly on how they created the crystal structure for the RPE65 complex and determined its active site binding activity.

      As far as an alternative pathway, one was discussed based on a previously reported RPE65 structure. Palmitate can bind to the inhibitor binding site if there aren’t any retinoids or inhibitors, and this is considered to be an off-pathway mode of binding palmitate. This binding allows palmitate to make 11-cis-retinal, which would be where the rod/cone regeneration comes in.

  4. Cool paper Nikki. It’s really impressive to me that these authors were able to solve the crystal structure for RPE65 let alone determine its mechanistic details since it is a membrane-bound structure. Those are pretty tricky to study. I’m curious as to why the authors felt the need to use two different “retinoid-like” molecules (emixustat and EB-001) as a part of their experimentation. Why not use only one of them or find yet another hybrid of emixustat to use? How could it have made their argument more convincing or this paper more impactful? Nice job summarizing!

    1. Yeah, membrane-bound proteins are very hard to crystallize which is why I was impressed with the authors and chose this paper! And thanks! I tried to summarize as best as I could with so much information to share! ^u^

      The authors don’t really explain why they decided to use two different “retinoid-like” molecules. MB-001 came about because they decided to enhance the structural similarity of emixustat, which made it a hybrid. They substituted a beta-ionone ring for the cyclohexyl moiety of emixustat. My best guess is that they wanted to see if they could improve upon an already known “retinoid-like” molecule. Maybe the authors thought this would make their paper more impactful if they could enhance an already good molecule for therapeutic purposes.

  5. Great paper! I thought their approach of using these potential drugs such as, emixustat and MB-001, that a binds to RBE65 at the active site to be very clever. After they provided evidence that these inhibitors acted at the active site of RPE65, which enabled them to to identify the amino acids involved and the role of the catalytic iron and palminate. I think one of the more broad contribution of this research was that this was “the first genuine example of of CCO-lagand complexes.” Which means that using similar methods more structural information about other carotenoid cleavage oxtygenases (CCO) can be obtained. Are you familiar with any other CCO’s vital to different pathways?

    1. Thanks Zach!

      Personally, I am not familiar with any other CCOs vital to different pathways. I did a little be of extra research to see what I could find. It seems as though there are only a handful of human proteins containing CCOs, and they all seem to be involved in the retinoid cycle pathway. Some plants and bacteria also have CCOs in some of their pathways. I hope that answered your question!

  6. Hey Nicole, a very interesting article and blog! As someone very interested in protein structure-function relationships, I found this to be a fascinating study. Since humans are more visual creatures than any other sense, the study of RPE65 is important in order to understand the mechanism and how the receptor functions. The article proposes a possible mechanism for RPE65 which includes a long list of reactions. Do the authors conduct any site direct mutagenesis studies to pinpoint essential residues or propose any residues they deem as vital to the receptor’s mechanism?

Comments are closed.