ABSTRACTCryptophyte algae are among the few eukaryotes that employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to the cyanobacterial PBP that are organized in large membrane-associated super complexes, the phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin PE545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. Thus far, the assembly of cryptophyte PBPs is not yet completely understood but involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of eukaryotic S-type PBP lyase GtCPES from G. theta. We show GtCPES mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP β-subunit (PmCpeB) of Prochlorococcus marinus MED4. Based on the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB) likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colorful complex in vitro and in vivo produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate hand over to the target protein.
Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity
