Identification of the key residues determining the product specificity of isomerohydrolase
- Authors
- Takahashi, Y., Moiseyev, G., Nikolaeva, O., and Ma, J.X.
- ID
- ZDB-PUB-120423-1
- Date
- 2012
- Source
- Biochemistry 51(20): 4217-4225 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Amino Acid Sequence
- Pigment Epithelium of Eye/metabolism
- Animals
- Molecular Sequence Data
- Substrate Specificity
- Chickens
- Phenylalanine/chemistry
- Tyrosine/chemistry
- Leucine/chemistry
- cis-trans-Isomerases/chemistry*
- cis-trans-Isomerases/genetics
- Vitamin A/metabolism
- Catalytic Domain*
- Zebrafish/metabolism*
- Mutagenesis, Site-Directed
- PubMed
- 22512451 Full text @ Biochemistry
The efficient recycling of the chromophore of visual pigments, 11-cis retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis retinol (11cROL) and a minor amount of 13-cis retinol (13cROL), from all-trans retinyl ester (atRE). We recently identified and characterized novel homologs of RPE65, RPE65c and 13-cis isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologs share 97% amino acid sequence identity, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103 and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. Particularly, our results showed that residue 58 is a primary determinant of isomerization specificity, since the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.