Identification of key residues determining isomerohydrolase activity of human RPE65 [Protein Structure and Folding]

August 11th, 2014 by Takahashi, Y., Moiseyev, G., Ma, J.-x.

RPE65 is the retinoid isomerohydrolase that converts all-trans retinyl ester to 11-cis retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65), but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian RPE65 and RPE65 from other lower vertebrates, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65 and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant N170K/K297G of hRPE65 exhibited significantly higher catalytic activity than that of wt hRPE65. Further, when an amino terminal fragment (1-33) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.