The role of Drosophila ninaG oxidoreductase in visual pigment chromophore biogenesis

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.