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Y. Takahashi, G. Moiseyev, Y. Chen, O. Nikolaeva, K. Farjo, M. Jian-xing; Identification and Characterization of a Novel 13-cis Isomerohydrolase. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1303. doi: https://doi.org/.
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13-cis Retinoic acid (13cRA), one of the stereoisomeric forms of retinoic acid (RA), is administrated to treat pathological conditions such as acute promyelocytic leukaemia, some skin diseases and tumors. Moreover, it has been shown that 13cRA has significant inhibitory effects on retinoid visual cycle. Although 13cRA is naturally occurring in the body, the mechanisms for its generation are unknown. We hypothesize that enzymatic generation of 13-cis retinol (13cROL) is the first and critical step in the generation of 13cRA. The purposes of this study were to identify and characterize the 13-cis isomerohydrolase (13cIMH) and identify the key residues determining the product specificity.
A candidate gene was cloned into an expression plasmid and transfected into 293A cells. The expression levels were confirmed by Western blot analysis and semi-quantified by densitometry. The enzymatic activities were measured by in vitro isomerohydrolase activity assay, and the generated retinoids were analyzed by HPLC. Further, we examined the substrate specificity, iron-dependency and subcellular fractionation of 13cIMH. In order to identify the key residues determining the specificity of products, candidate residues were mutated by site-directed mutagenesis. The mutants of 13cIMH were expressed, and their products were analyzed by in vitro isomerohydrolase activity assay.
A novel protein of a calculated molecular weight of 60567Da was identified to generate exclusively 13cROL from all-trans retinyl ester, but not from all-trans retinol, and the isomerohydrolase activity of 13cIMH was dependent on iron. The 13cIMH expressed in 293A cells was present in both the membrane and cytosol fractions. RT-PCR showed that this enzyme is expressed in both the brain and eye. A single point mutation L103F in 13cIMH shifted the product from exclusively 13cROL to both 13cROL and 11cROL.
A 13cIMH is the firstly identified enzyme to specifically generate 13cROL in the brain and eye, which may contribute to the generation of endogenous 13cRA.
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