Reconstitution of Blue−Green Reversible Photoconversion of a Cyanobacterial Photoreceptor, PixJ1, in Phycocyanobilin-Producing Escherichia coli

Abstract

PixJ1, a photoreceptor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, mediates positive phototactic motility and contains two GAF domains, the latter of which binds a bilin chromophore. Full-length PixJ1 expressed and purified from Synechocystis showed unique reversible photoconversion between a blue light-absorbing (Pb) form and a green light-absorbing (Pg) form (1) in contrast to the reversible phototransformation between the red light-absorbing form and far-red light-absorbing form of the other GAF-containing photoreceptors such as plant or bacterial phytochromes. To clarify the origin of the blue-shifted photoconversion, we tried to reconstitute this blue-green reversible phototransformation by synthesizing the second GAF domain in Escherichia coli transformed with genes for biosynthesis of four different bilins, biliverdin (BV), bilirubin (BR), phycocyanobilin (PCB), and phycocyanorubin (PCR), as final products. The three expression systems, the BR system being the exception, produced a GAF polypeptide with a covalently bound bilin. The GAF polypeptide from the BV-synthesizing system exhibited an irreversible photoconversion, while that from the PCB-synthesizing system revealed photoconversion between Pb and Pg almost identical to that of the full-length PixJ1, indicating that PCB is responsible for the blue-green reversible photoconversion. Furthermore, the GAF polypeptide from the PCR-producing system exhibited almost the same reversible spectral change, possibly coming from the PCB accumulated in the PCR-biosynthetic pathway. Mass spectrometry (MS) of the main tryptic chromopeptide revealed that the chromophore binds to a 21-amino acid peptide that contains a cysteine-histidine motif for phytochrome chromophore binding and that an ion signal can be assigned to desorbed PCB. The absorption spectra of the denatured GAF polypeptide suggested that PCB is attached to the protein moiety in a twisted conformation that disrupts the pi-electron conjugation between the A and B rings, possibly being held in position through a second covalent linkage.