Studies On The Biosynthesis Of Phycocyanin And Allophycocyanin From Anabaena

Cyanobacteria is the photosynthesis microorganism. Under the fitting water environment, a great deal of algae can grown which can induced water bloom. Phycobiliproteins, a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes, are the function composition of light-harvesting complexes in the algae photosynthesis. The last step in phycobiliprotein biosynthesis is the phycobilin addition to the apophycobiliproteins. In vivo, the correct attachment of most chromophores is catalyzed by lyases, of which only few have been characterized so far. As we know now, PecE and PecF catalyzed both the attachment and isomerization of phycocyanobilin to PecA, while the covalent attachment of phycocyanobilin to theα-subunit of C-phycocyanin, CpcA, is catalyzed by CpcE and CpcF. But heretofore nothing is reported about the biosynthesis of the core-membrane linker protein (LCM), theβ-subunits of C-phycocyanin and C-phycoerythrocyanin (CpcB and PecB), and the subunits of allophycocyanin (APC).In this paper, the biosynthesis of LCM, CpcB, PecB and the five subumits of APC from Anabaena sp. PCC7120 was studied by the in vitro and in vivo reconstitution.ApcE and C-terminally truncated ApcE(1-240) bind covalently phycocyanobilin (PCB) in an autocatalytic reaction were firstly studied on its cofactor in the reconstitution system in vitro. Mercaptoethanol (ME) is necessary to the reconstitution, 4mol/L urea increased the reconstitution yield and the addition of sucrose is useful for this reaction. The optimal reaction system is 20mmol/L potassium phosphate buffer (KPB, pH 7.2) containing 100mmol/L NaCl, 4mol/L urea, 200mmol/L sucrose, 0.1mmol/L ME. After purified by the Ni2+-affinity chromatography, LCM(1-240) had absorption at 661 nm and fluorescence at 672 nm. The CD extrema (662, 604, 383 and 347 nm) match the absorption maxima. This and the absorption ration (QAvis/uv =4.1), the fluorescence quantum yield (Φf=0.43), the extinction coefficient (ε=93000 M-1 cm-1) suggest that PCB in the reconstituted products is in a near native chromophore conformation of LCM . The kinetic analyses of the reaction of PCB with ApcE(1-240) have also studied, and giving the following parameters: Km=1.3μmol/L,Vmax=9.6 nmol/L·s-1,kcat=9.6×10-4 s-1. It's also verified by means of acid-urea denaturation and UV-induced fluorescence by Zn2+-SDS gels tests.CpcB(C155I) and PecB(C155I) bind covalently PCB in vitro and in vivo in E. coli catalyzed by CpeS were firstly studied. The apo-proteins CpcB(C155I) (PecB(C155I), CpcB, PecB), the lyase CpeS, heme oxygenase 1 (HO1) and PCB:ferredoxin oxidoreductase (PcyA) were co-expressed in E. coli by a dual vector system. Reconstitution chromoproteins, purified by Ni2+-affinity chromatography had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629nm respectively. After denaturation in acidic urea solution (8mol/L, pH 2.0), reconstituted chromoproteins gave maximal absorption at 662 nm. Reconstituted and purified PCB-CpcB(C155I) and PCB-PecB(C155I) were analyzed via SDS-PAGE: in the presence of Zn2+ they showed the fluorescence that is characteristic for bilins covalently bound to the proteins. We have got the the absorption ration, the fluorescence quantum yield and the extinction coefficient on the base of the experimental spectra. Purified PCB-CpcB(C155I) and PCB-PecB(C155I) biosynthesized in E. coli were finally digested with pepsin under acidic conditions. They have the same maximal absorption and HPLC analysis as the nativeβ-PEC andβ-CPC. PCB also can attach to CpcB and PecB in vitro reconstitution, and the maximal absorption and fluorescence has a 2-3nm red shift compared to the chromoproteins biosynthesized in E. coli. All these declared that CpeS catalyzes the site-selective attachment of PCB to cysteine-β84 in both CpcB and PecB.The five subunits (ApcA, ApcB, ApcA2, ApcD, ApcF) of allophycocyanin bind covalently PCB in vivo in E. coli catalyzed by CpeS were firstly studied. The apo-protein ApcA (ApcB, ApcA2, ApcD, ApcF), the lyase CpeS, heme oxygenase 1 (HO1) and PCB:ferredoxin oxidoreductase (PcyA) were co-expressed in E. coli by a dual vector system. Reconstitution chromoproteins, purified by Ni2+-affinity chromatography had absorption maxima and fluorescence emission maxima at 618 nm/642 nm, 612 nm/640 nm, 622 nm/642 nm, 650 nm/663 nm (602 nm/635 nm) and 624 nm/644 nm respectively. After denaturation in acidic urea solution (8mol/L, pH 2.0), reconstituted chromoproteins gave maximal absorption at 662-664 nm. Reconstituted and purified PCB-ApcA , PCB-ApcB, PCB-ApcA2, PCB-ApcD, PCB-ApcF were analyzed via SDS-PAGE: in the presence of Zn2+ they showed the fluorescence that is characteristic for bilins covalently bound to the proteins. We have got the the absorption ration, the fluorescence quantum yield and the extinction coefficient on the base of the experimental spectra. Purified PCB-ApcA and PCB-ApcB biosynthesized in E. coli were finally digested with pepsin under acidic conditions. They have the analogical maximal absorption and HPLC analysis as the native APC. All these and chromoproteins mass spectrum analysis declared that CpeS catalyzes the attachment of PCB to Cys-82 in these five APC subunits.We also have done researchs on the reconstitution of the up five APC subunits of allophycocyanin bind covalently PCB in vitro catalyzed by CpeS. CpeS only can catalyze PCB bind to ApcA, ApcD and ApcF. The cofactors in the reconstitution system and the kinetic analysis have been studied. Potassium phosphate buffer (KPB, pH 7.2) is the opatimal buffer for the reconstitution. The suitable concentration of NaCl is between 100-200mmol/L. 37℃is the proper reconstitution reaction temperature. Mg2+ can enhance the efficiency of PCB-ApcA reconstituted in vitro, but it have not influence on the PCB-ApcD and PCB-ApcF reconstituted in vitro. Mercaptoethanol (ME) has great influence on the spectra form of PCB-ApcD. Other metal ions restrain the reconstitution efficiency. And the other cofactors have not any influence. So we did not add any cofactors in the reconstitution of ApcD and ApcF. The kinetic analysis indicated that these values agree well with the range obtained for PCB attachment to cysteine-84 of CPC and PEC by the E/F-type lyases and CpeS. Purified PCB-ApcA, PCB-ApcD and PCB-ApcF, produced in vitro from the respective apoproteins and PCB under the action of CpeS without His-tag, had the same spectra and spectral parameters as biosynthesized chromoproteins in E. coli. These also show that only a single PCB had been covalently bound in vitro to Cys-82 of ApcA, ApcD and ApcF, respectively.