Abstract
Allophycocyanin (APC), a protein carrying tetrapyrrole phycocyanobilin (PCB), performs distinctive absorption and emission in visible light range. Recombinant APC alpha subunit (ApcA) was shown to be a monomeric protein by analytic ultracentrifugation and size-exclusion chromatography. ApcA is an autocatalytic bilin:protein lyase to facilitate the biosynthesis of functional chromophorylated ApcA (chromo-ApcA). In order to investigate the critical residues in the lyase active site of ApcA, thirteen residues around PCB were mutated by site-directed mutagenesis. Mutants R83A and D84A abolished lyase activity although there was no difference in secondary structure between mutants and wild-type ApcA. Lower fluorescence intensity of other mutants was observed in comparison with wild type despite these mutants still kept the ability of PCB attachment to protein. Complete loss of lyase activity in two mutants, Arg83 and Asp84, perhaps resulted from the interference of hydrogen bonding interaction with PCB. In addition, residues which are located around the active site and involved in the hydrophobic interaction with PCB affect the fluorescence intensity of chromophorylated proteins.