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Y. Takahashi, G. Moiseyev, J.-X. Ma; Key Residues of RDH10 (Retinol Dehydrogenase Class 10) for Its Enzymatic Activity and Membrane Association. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3253.
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© ARVO (1962-2015); The Authors (2016-present)
RDH10 is an enzyme identified in retinal pigment epithelium (RPE) and retinal Müller cells and catalyzes oxidation of all-trans retinol to all-trans retinal in an NADP-dependent manner. Amino acid sequence of RDH10 is highly conserved across species, suggesting its functional significance. The purpose of this study was to define key residues in the catalytic center, cofactor-binding motif and transmembrane (or membrane association) domains.
Wild-type (wt) human RDH10 and its mutants were transiently expressed in COS-1 cells. Expression of recombinant RDH10 was confirmed by Western blot analyses. The enzymatic activity was measured by in vitro activity assay using isolated total membrane proteins and analyzed by HPLC. Two hydrophobic regions (Δ2-23 or Δ293-329), potential transmembrane or membrane association domains, were deleted. The subcellular localizations of the truncated mutants were analyzed by subcellular fractionation.
Total membrane proteins from COS-1 cells expressing wt human RDH10 showed significant enzymatic activities converting all-trans retinol to all-trans retinal. Substitution of any of amino acid residues in predicted catalytic sites did not significantly affect protein levels of RDH10, but most of single mutations, except for mutation at Ser197, abolished the enzymatic activity. Mutant Ser197Ala retained lower than that of wt but significant enzymatic activity whereas Ser197Thr showed no detectable enzymatic activity. Substitution of all three glycine residues in cofactor-binding motif (Gly-X3-Gly-X-Gly) abolished enzymatic activity, probably due to loss of the cofactor bound to the enzyme. The mutant with deleted N-terminal hydrophobic domain did not show detectable activity and was dissociated from the membrane.
The molecular model of RDH10 is predicted based on 17b-hydroxysteroid dehydrogenase crystal structure. Predicted RDH10 structure model possesses a similar catalytic center, even though it shares a low sequence identity with other members of the short-chain dehydrogenase/reductase family, including other RDHs. The key residues in the predicted catalytic center and cofactor-binding motif are essential for its enzymatic activity. Membrane association seems to be essential for its enzymatic activity as well.
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