AppA, a redox regulator of photosystem formation in Rhodobacter sphaeroides 2.4.1, is a flavoprotein - Identification of a novel FAD binding domain

The AppA protein is required for increased photosystem gene expression upon transition of the facultatively photoheterotrophic bacterium Rhodobacter s phaeroides 2.4.1 from aerobic to anaerobic photosynthetic conditions. AppA shows no obvious similarity to proteins with established function. Genetic evidence suggests that its effect is exerted through modulation of the acti vity of the repressor PpsR, which controls expression of multiple photosyst em genes. To gain insight into the nature of AppA involvement in redox-depe ndent photosystem gene expression, the appA gene was overexpressed in Esche richia coli, AppA was produced as insoluble inclusion bodies. The purified inclusion bodies were found to contain FAD, By overexpressing various delet ion derivatives, we were able to localize the region of AppA sufficient for FAD binding to approximately 120 aminoterminal residues. To assess the rol e of FAD binding in AppA function, we constructed an AppA derivative lackin g the entire FAD binding domain. Surprisingly, this derivative complemented the AppA null mutant undergoing transition from aerobic to anaerobic photo synthetic growth conditions almost to the same extent as the full-length Ap pA protein. When the sequence of the amino-terminal portion of AppA was exa mined, it was shown not to contain any known flavin binding motifs, However , two open reading frames of unknown function, showing significant similari ty to the amino terminus of AppA, were identified, i.e. Synechocystis sp, S r11694 and E. coli F403, The latter gene was amplified and overexpressed in E, coli, and the partially purified F403 protein was found to contain FAD as a cofactor. We have therefore concluded that the amino terminus of AppA represents a novel FAD binding domain present in a small group of bacterial proteins. The binding of FAD by AppA may be the first clue as to how this regulatory protein is involved in redox-regulated reactions.