| Cyanobacteria are the most primitive organism which produce oxygen during photosynthesis. Because of cyanobacteria, the atmosphere of the earth changes from oxygen-deprived to aerobic environment. However, we couldn’t imagine how harmful cyanobacteria algal blooms. Since the industrial revolution, algal blooms pose an increasing serious water pollution problem all over the world. It will give rise to water degradation, and the deaths of fish. And worst of all, a microcytis bloom will bring about microcystin, which is the main precipitating factor of liver cancer. That looking for an effective inhibitor is the only way to control cyanobacteria invasion.Specific target of cyanobacteria may provide useful solutions to the development of algicide. Fructose-1,6-bisphosphatases from cyanobacteria(Cy-FBP/SBPase) is an essential regulatory enzyme in the Calvin cycle. Previous studies have demonstrated that the disruption of FBPase gene is lethal to cyanobacteria. Moreover, FBPase content is less than 1% of the whole protein.That’s to say, Cy-FBPase will be a potential target inducing the death of cyanobacteria. We choose to study Cy-FBPase in hope of further research on it. We mainly carried out the word in following five areas:First, we constructed the plasmid of Cy-FBP/SBPase and established the expression and purification systems.Second, based on the evaluation of the enzyme assay methods which had been reported, we developed a phosphate color-test method for Cy-FBP/SBPase assay using 96-well plate. It’s better for high throughput screening.Third, we tried to study the enzymatic property and structure of Cy-FBP/SBPase through the following areas:1. The crystal structure of Cy-FBPase-AMP was solved at 2.3 A resolution and Cy-FBPase-AMP-FBP was solved at 2.4 A, respectively. Cy-FBPase is a homotetramer, the same as FBPase from pig liver or pea chloroplast.2. To identify the active site residues involved in FBP hydrolysis, we have mutated 13 residues of Cy-FBPase to alanine or glycine. In addition,6 residues in AMP binding sites are mutated in order to get important information of the structure.3. Comparing to FBPase from pig liver, there are differences in FBP and AMP binding sites. Obviously that provides important guidance to our work.Forth, we studied regulation function of Cy-FBP/SBPase, the work was carried out as following:1.The interaction between Cy-FBP/SBPase and metal ions were studied by enzyme activity analysis, fluorescence spectrum, gel filtration and isothermal titration calorimetry. Cy-FBP/SBPase can be activated by Mg2+, Mn2+, but can not be activated by Ca2+or Zn2+. Metal cations were stabilizing the quaternary conformation. Spectroscopic analysis of emission quenching showed that quenching mechanism of Cy-FBP/SBPase with Mg2+or Mn2+was static quenching mechanism while that of Ca2+ or Zn2+ was dynamic quenching process. Divalent metal ions will change Cy-FBPase tertiary structure while second structure remains the same.2. For the wild type Cy-FBP/SBPase, the binding of AMP alone can drive the relax to tense state transition, in which the upper subunit -pair rotates 14°relative to the bottom subunit -pair of the FBPase tetramer. The interactions between Cy-FBP/SBPase and AMP are entropically (-TΔS=-1245cal·mor-1) and enthalpically(ΔH=-5950 cal·mol-1) driven, but not with equal contributions. In the conditions of Mg2+, AMP generally exhibits the greatest enthalpy contributions (ΔH=-12100 cal·mol-1). In the presence of AMP, Cy-FBP/SBPase changes from dimmer to tetramer. In the whole process, second structure was still unchanged, but tertiary structure changed a lot by comparison.3. Cy-FBPase is activated in the light by changes in the tertiary structure of the enzyme induced by reduction via thethioredoxin/ferredoxin system. In vivo, we use DL-dithiothreitol (DTT) instead of thethioredoxin/ferredoxin system. Cys75 and Cys84 form a disulfide bridge in Cy-FBP/SBPase-AMP complex structure. However, C84S was sensitive to DTT, high Mg2+ levels, or thioredoxin activation and behaved as wild type enzyme, while C75S was insensitive to all above. That is, Cys84 has not formed a disulfide bridge with Cys75. In fact, Cys75 and Cys99 are partners of the disulfide bridge, while the two cysteines are at a distance of 28.32 A in crystal structure. Therefore, a homotetramer is not just assembling, but also undergoes a structure change when dimmer to tetramer.Fifth, based on the structure and properties of Cy-FBP/SBPase, we are sure that there are at least three states:initial state(I-state), relax state(R-state), and tense state(T-state). I-state means Cy-FBP/SBPase shows dimmer structure without ligand and Cys75 and Cys99 form a disulfide bridge. R-state means metal cations bind to Cy-FBP/SBPase. In the presence of AMP, Cy-FBP/SBPase is a homotetramer. The three states can change into another state under certain conditions. And then we proposed catalytic mechanism of Cy-FBP/SBPase. One of the two metals is responsible for coordinating the phosphoester bond and stabilizing the negative charge on the leaving group, whereas the second metal ion coordinates the nucleophilic water. The hydrogen bonding with the nearby threonine and carboxylate (Asp or Glu) residues activate this water molecular.In summery, this paper established the systems of expression, purification, enzyme assay and site-directed mutation of Cy-FBP/SBPase. The structural biology research was also carried out. The interactions between the enzyme and AMP or metal cations were studied by fluorescence spectrum, gel filtration and isothermal titration calorimetry. All these results would shed light on the research of regulate function, as well as catalytic mechanism. |
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