A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70-complexed and competent for flavinylation, membrane association and enzymatic activation

Abstract

A cDNA coding for the carotenoid biosynthetic enzyme phytoene desaturase from Narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane-bound populations of phytoene desaturase in the chromoplasts isolated from petals. The soluble form is enzymatically inactive and a constituent of a larger Hsp 70-containing protein complex in the stroma, whereas the membrane-bound form is functional. In vitro, the soluble form is able to associate on to/into protein-free liposomal membranes made from chromoplast lipids, thereby gaining activity by binding added flavine adenine dinucleotide (FAD). Once bound to membranes, activated phytoene desaturase works independently of any added FAD, employing membrane-bound electron acceptors. FAD, however, exerts no positive effect on the membrane-association process. Its role is confined to enzymatic activation. Although carotenoid accumulation is strongly induced during flower development, only very low concentrations of phytoene desaturase transcripts are detectable, while the corresponding protein accumulates in low, but measurable amounts, appearing in soluble and membrane-bound states. Post-transcriptional mechanisms contribute significantly to carotenoid accumulation, as do factors determining the enzymatic activity of phytoene desaturase, for example by influencing the redox-state of membrane-bound electron acceptors.