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Yusuke Takahashi, Gennadiy P. Moiseyev, Olga Nikolaeva, Jian-xing Ma; Identification of The Key Residues Determining The Product Specificity of Isomerohydrolase. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3355.
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© ARVO (1962-2015); The Authors (2016-present)
The efficient recycling of the visual pigment chromophore, 11-cis retinal, through the retinoid visual cycle is essential for maintaining normal vision. RPE65 is the isomerohydrolase which catalyzes the key reaction which generates predominant 11-cis retinol (11cROL) and a minor amount of 13-cis retinol (13cROL), from all-trans retinyl ester. The molecular mechanisms and the key residues that determine the isomerization specificity of RPE65 have not been well-defined. The purpose of this study is to identify the key residues that determine isomerization specificity of RPE65.
We recently identified and characterized two novel homologs of RPE65, RPE65c and 13-cis isomerohydrolase (13cIMH), which are expressed in the zebrafish retina and brain, respectively. Although these two isoforms shared 97% of amino acid sequence identify, RPE65c generated predominantly 11cROL, whereas 13cIMH generated exclusively 13cROL in the in vitro assay system. To identify the key residues which confer the product specificity of these two enzymes, we focused on the divergent residues in RPE65c and 13cIMH and replaced these candidate residues by site-directed mutagenesis. Expression levels of the mutants were confirmed by Western blot analysis. The enzymatic activities were determined by performing in vitro isomerohydrolase activity assays using HPLC analysis and quantification of produced retinoids.
Single point mutations in RPE65c, Y58N, F103L and L133S altered the isomerization specificity of RPE65c, increasing 13cROL while decreasing 11cROL production, compared to that of wild-type RPE65c. On the other hand, a mutation N58Y in 13cIMH completely switched the isomerization specificity to that of RPE65c.
These results suggest that residue 58 is a primary key residue that determines isomerization specificity in the novel homologs of zebrafish RPE65. Identification of key residues determining the product specificity will contribute to the understanding of mechanism for isomerization reaction catalyzed by RPE65.
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