The RPE65 gene (UniProtKB/Swiss-Prot: Q16518 (RPE65_Human); OMIM: 180069; http://www. ncbi. nlm. nih. gov/entrez/dispomim. cgi? id= 180069) is localized to human chromosome 1p31 and to distal mouse chromosome 3 and is a single copy gene, except in zebrafish where there are 2 copies, a consequence of the genome duplication in the teleost ancestry. RPE65 protein is highly preferentially expressed in retinal pigment epithelium (RPE) of all vertebrates (Fig. 1A), and may be expressed at low level in cone photoreceptors of some species. All RPE65 proteins are 533 amino acid residues in length, except in zebrafish where RPE65a gene product has 531 and RPE65b gene product 532 residues, respectively. RPE65 belongs to the evolutionarily diverse carotenoid oxygenase superfamily. The basic reaction catalyzed by these enzymes is oxidative cleavage of carbon-carbon double bonds in the polyene backbone of carotenoids and related molecules. One of RPE65’s 2 mammalian relatives is βcarotene monooxygenase, which cleaves β-carotene into all-trans retinal, the first step in animal vitamin A metabolism. The crystal structure of cyanobacterial Synechocystis apocarotenal oxygenase, a carotenoid oxygenase, predicts that these proteins share a common seven-bladed propeller structure containing non-heme ferrous iron coordinated by four histidines, three of which are fixed by glutamate residues. Each blade of the propeller, consisting of 4 or 5 anti-parallel beta-sheets, contributes one part of the iron coordination apparatus (Fig. 1B). The locations of these beta sheets are conserved and these contribute to a rigid structure. Inter-strand and inter-blade loops are less conserved and are predicted to contribute to specificity of substrate binding in the different family members. RPE65 is predicted to share the overall secondary structure required to support this tertiary structure and mutagenesis studies support the requirement of the coordinating histidines in RPE65 activity