Orange protein, phytoene synthase regulator, has protein disulfide reductase activity

Orange protein (OR) is known to interact with phytoene synthase (PSY) that commits the first step in carotenoid biosynthesis, and functions as a major post-transcriptional regulator on PSY. We here tried to reveal enzymatic characteristics of OR, that is, protein disulfide reductase (PDR) activity of the Arabidopsis thaliana OR protein (AtOR) was analyzed using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The AtOR part containing only the zinc (Zn)-finger motif was found to show PDR activity, with an apparent K_m of 12,632 nM, K_cat of 11.85 min^-1, and K_catK_m^-1 of 15.6 × 10³ M^-1sec^-1. To evaluate the significance of the N-terminal region of AtOR, we examined the kinetic parameters of a fusion protein composed of the N-terminal region and the Zn-finger motif from AtOR. Consequently, the fusion protein had lower values for K_m (2,074 nM) and K_cat (3.18 min^-1) and higher catalytic efficiency (25.9 × 10³ M^-1sec^-1) than that of only the Zn-finger motif part, suggesting that the N-terminal region of AtOR should be important for substrate affinity and catalytic efficiency of PDR activity. Complementation experiments with E. coli further demonstrated that AtOR containing the N-terminal region and the Zn-finger motif increases phytoene synthase activity of AtPSY especially under reduced circumstances retaining a NADPH- and H⁺-regeneration system.