Structural And Functional Studies Of Cyanobacteria Light-dependent Protochlorophyllide Oxidoreductase

The organic substances produced by photosynthesis are the energy source for plant,animal and human life activities.Chlorophyll is the main pigment for photosynthesis and is responsible for the absorption,conversion and transmission of solar energy.The biosynthesis of chlorophyll is an extremely complex biological reaction process,which begins with 5-aminolevulinic acid and through eight steps of enzymatic reaction forms a key compound in the tetrapyrrole biosynthetic pathway,protoporphyrin IX.Protoporphyrin IX differentiates into two branches: the chlorophyll branch and the heme branch.Following 7-step enzymatic reactions initiated by magnesium chelation into protoporphyrin IX by magnesium chelatase,the final product is chlorophyll a.Light-dependent protochlorophyllide chlorooxidase(LPOR,EC 1.3.1.33)plays a key role in the chlorophyll branch.It catalyzes the penultimate step,which requires light and the coenzyme NADPH.The C17=C18 double bond in the substrate protochlorophyllide is reduced to yield the product chlorophyllide.In the thesis,we studied the structure and catalytic mechanism of LPOR from two photosynthetic bacteria,Synechocystis sp.PCC 6803 and Thermosynechococcus elongatus(SyLPOR and TeLPOR).We reconstructed the recombinant plasmids encoding the target gene,transformed them respectively into Escherichia coli cells for heterogenetic expression.The recombinant protein was purified by nickel affinity chromatography followed by gel filtration chromatography.Stable SyLPOR and TeLPOR with high purity were obtained and crystallized using vapor diffusion method.The crystals of NADPH-bound LPOR were diffracted to 2.2 ?(SyLPOR)and 2.4 ?(TeLPOR).The two structures were determined by molecular replacement.The results revealed that there are 2 protomers in an asymmetric unit and each protomer binds to one NADPH molecule.The substrate pocket lies near the nicotinamide moiety of NADPH.The two structures are highly similar except the regions containing the helix G,which shows significant difference.This indicated that helix G can undergo large conformational change upon substrate binding.In addition,the structures also suggested the proton-relay path during catalysis.