title: Identification of the key residues determining the product specificity of isomerohydrolase.
abstract: 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 homologues of RPE65, RPE65c, and 13-cis-isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologues have 97% identical amino acid sequences, 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 the 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. In particular, our results showed that residue 58 is a primary determinant of isomerization specificity, because 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.
authors.affiliation: Department of Medicine Endocrinology, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Gennadiy
authors.initials: G
authors.lastname: Moiseyev
authors.forename: Olga
authors.initials: O
authors.lastname: Nikolaeva
authors.forename: Jian-xing
authors.initials: JX
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002645:Chickens; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry; D014801:Vitamin A / Q000378:metabolism; D015027:Zebrafish / Q000378:metabolism*; D019745:cis-trans-Isomerases / Q000737:chemistry* / Q000235:genetics; D000099178:Retinoid Isomerohydrolase
chemical_list: D007930:Leucine; D010649:Phenylalanine; D014443:Tyrosine; D014801:Vitamin A; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase