AppA, a redox regulator of photosystem formation in Rhodobacter sphaeroides 2.4.1, is a flavoprotein. Identification...

The AppA protein is required for increased photosystem gene expression upon transition of the facultatively photoheterotrophic bacterium Rhodobacter sphaeroides 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 activity of the repressor PpsR, which controls expression of multiple photosystem genes. To gain insight into the nature of AppA involvement in redox-dependent photosystem gene expression, the appA gene was overexpressed in Escherichia coli. AppA was produced as insoluble inclusion bodies. The purified inclusion bodies were found to contain FAD. By overexpressing various deletion derivatives, we were able to localize the region of AppA sufficient for FAD binding to approximately 120 amino-terminal residues. To assess the role of FAD binding in AppA function, we constructed an AppA derivative lacking the entire FAD binding domain. Surprisingly, this derivative complemented the AppA null mutant undergoing transition from aerobic to anaerobic photosynthetic growth conditions almost to the same extent as the full-length AppA protein. When the sequence of the amino-terminal portion of AppA was examined, it was shown not to contain any known flavin binding motifs. However, two open reading frames of unknown function, showing significant similarity to the amino terminus of AppA, were identified, i.e. Synechocystis sp. Srl1694 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.