Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides

The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL). This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch.