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

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• 作者：Shizue Yoshihara ; Takashi Shimada ; Daisuke Matsuoka ; Kazunori Zikihara ; Takayuki Kohchi ; and Satoru Tokutomi
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：March 21, 2006
• 年：2006
• 卷：45
• 期：11
• 页码：3775 - 3784
• 全文大小：277K
• 年卷期：v.45,no.11(March 21, 2006)
• ISSN：1520-4995

文摘

PixJ1, a photoreceptor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, mediatespositive 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 photoconversionbetween a blue light-absorbing (Pb) form and a green light-absorbing (Pg) form (1) in contrast to thereversible phototransformation between the red light-absorbing form and far-red light-absorbing form ofthe other GAF-containing photoreceptors such as plant or bacterial phytochromes. To clarify the origin ofthe blue-shifted photoconversion, we tried to reconstitute this blue-green reversible phototransformationby synthesizing the second GAF domain in Escherichia coli transformed with genes for biosynthesis offour 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 GAFpolypeptide with a covalently bound bilin. The GAF polypeptide from the BV-synthesizing system exhibitedan irreversible photoconversion, while that from the PCB-synthesizing system revealed photoconversionbetween Pb and Pg almost identical to that of the full-length PixJ1, indicating that PCB is responsible forthe blue-green reversible photoconversion. Furthermore, the GAF polypeptide from the PCR-producingsystem exhibited almost the same reversible spectral change, possibly coming from the PCB accumulatedin the PCR-biosynthetic pathway. Mass spectrometry (MS) of the main tryptic chromopeptide revealedthat the chromophore binds to a 21-amino acid peptide that contains a cysteine-histidine motif forphytochrome chromophore binding and that an ion signal can be assigned to desorbed PCB. The absorptionspectra of the denatured GAF polypeptide suggested that PCB is attached to the protein moiety in atwisted conformation that disrupts the -electron conjugation between the A and B rings, possibly beingheld in position through a second covalent linkage.