Identification of significant residues for intermediate accumulation in phycocyanobilin synthesis.

Phycocyanobilin, the primary pigment of both light perception and light-harvesting in cyanobacteria, is synthesized from biliverdin IXα (BV) through intermediate 181, 182-dihydrobiliverdin (181, 182-DHBV) by a phycocyanobilin:ferredoxin oxidoreductase (PcyA). In our previous study, we discovered two PcyA homologs (AmPcyAc and AmPcyAp) derived from Acaryochloris marina MBIC 11017 (A. marina) that exceptionally uses chlorophyll d as the primary photosynthetic pigment, absorbing longer wavelength far-red light than chlorophyll a, the photosynthetic pigment found in most cyanobacteria. Biochemical characterization of the two PcyA homologs identified functional diversification of these two enzymes: AmPcyAc provides 181, 182-DHBV, and PCB to the cyanobacteriochrome (CBCR) photoreceptors, whereas, AmPcyAp specifically provides PCB to the light-harvesting phycobilisome subunit. In this study, we focused on the residues necessary for 181, 182-DHBV supply to the CBCR photoreceptors by AmPcyAc. Based on the SyPcyA structure, we concentrated on the 30 residues that constitute the substrate-binding pocket. Among them, we discovered that Leu151 and Val225 in AmPcyAc were both substituted with isoleucine. During the enzymatic reaction, the SyPcyA variant molecule, possessing V225I and L151I replacements, accumulates the 181, 182-DHBV and supplies it to a CBCR molecule derived from A. marina. It is worth noting that the substitution of Val225 with isoleucine was specifically conserved among the Acaryochloris genus. Collectively, we propose that the specific evolution of PcyA among the Acaryochloris genus may correlate with the acquisition of Chl. d synthetic ability and growth in long-wavelength far-red light environments.