Cyanobacteria Allophycocyanin The Biosynthesis And Assembly

Cyanobacterium is one of the primitive species with the capacity of oxygenic photosynthesis. The process of photosynthesis is initiated by the absorption of light energy through phycobiliproteins. Phycobiliproteins are multi-subunit complex that covalently bind a variety of linear tetrapyrolle pigments called bilins, enabling them to harvest light in the visible region of the spectrum. The absorbed energy can be transferred at almost 100% efficiency to the reaction center.Allophycocyanin (APC), located in the core of phycobilisome, has the special spectroscopic characteristics. APC is tightly associatedαβheterodimer, with only one bilin covalently attached to each subunit. One of the prominent spectroscopic characteristics of APC is the strong red-shift of the absorption and fluorescent emission maxima when monomers assembling into trimers. APC participates in the energy transfer in higher aggregates, i.e. APC trimer. Therefore, the researches on the biosynthesis and assembly of APC will provide insight into its function, i.e. the basis of the energy transfer. Further, these will also help us in the reconstitution of the giant complex phycobilisome.In this study, the entire pathway for the biosynthesis of holo-αandβsubunits of APC from Synechocystis sp. PCC 6803 was reconstituted in Escherichia coli (E. coli). The genes for apo-proteins (apo-ApcA or apo-ApcB) biosythesis, the genes ho1 and pcyA encoding the enzymes for PCB production and the genes cpeS-1 and cpcU for the attachment of PCB to apo-proteins were co-expressed in E. coli by a dual vector system. Upon induction, holo-αandβsubunits of APC were acquired with spectroscopic properties similar to those of the same protein produced in cyanobacteria. The results were also shown that CpcS/U is the bilin lyase which responsible for attachment of PCB to both ApcA and ApcB. In addition, the reconstitution of holo-ApcA was performed in vitro and the possible catalysis mechanism for the attachment of PCB to apo-ApcA was also suggested. The results demonstrated that CpcS/U can form a heterodimer so as to improve their solubility and stability. Lyases CpcS/U can bind rapidly to PCB in a non-covalent way, and transfer it to apo-ApcA slowly under the appropriated conditions. The reconstituted products have the characteristics identical to that of native APC holo-αsubunit. PCB can spontaneously attach to apo-ApcA in a much slower way, however, complex with correct absorption maxima could not be acquired in the absence of lyases CpcS/U. Both the reconstitution of APCsubunits in vivo in E. coli and in vitro will provide insight into the biosynthetic process of APC in cyanobacteria.In this study, the fluorescent APC trimer was successfully synthesized from Synechocystis sp. PCC 6803 in E. coli. Genes encoding APC apo-α,βsubunits and enzymes for phycocyanobilin (PCB) biosynthesis and covalent attachment to apo-proteins were co-expressed in E. coli using a dual plasmid system. The recombinant APC trimer (rAPC) was purified by using metal affinity and size exclusion chromatography and showed the characteristic absorption and emission spectra identical to those of the native APC, suggesting that rAPC was in its native conformation. Tryptic digestion analysis confirms the rAPC has the same components as the native APC. The molecular weight analysis (by HPLC) results clearly show that rAPC is in a trimeric state. This is the first study on the assembly of recombinant ApcA and ApcB to a trimer with the native structure.When compared to the study in which only monomer was formed, our results indicate that the synthesis of bilins and the subsequent attachment to apo-subunits are very important for the successful assembly of APC trimers.LCM is a multifunction linker serving as a terminal energy acceptor and a membrane anchor. Information on their biochemical properties and biosynthesis is scare due to their insolubility. In this study, the pathway for the synthesis of LCM(1-240) was reconstituted in E. coli. The recombinant LCM(1-240) is prone to aggregation as inclusive body. These insoluble products were dissolved in 4 M urea to acquire the spectroscopic properties similar to those of the reported proteins. The solubility of LCM(1-240) is improved when it is expressed as a MBP fusion, without the changes of the spectroscopic properties. This study will facilitate the biochemical analysis of LCM(1-240), and allow further understanding of their functions.Our studies focus on the biosynthesis and assembly of APC, which would help us to understand the relationship between the structure and the functions, reflecting on the change of its spectra. These would also facilitate the interaction analysis of multi-subunits of APC, which leads to the assembly of trimers. Additionally, as APC is a widely used fluorescent tags with numerous applications in biological and clinical fields, this study will provide a promising way to produce better fluorescent tags by molecular design.