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Open Fang SHEN Master Submit The Pennsylvania State University The Graduate School Intercollege Graduate Program in Plant Biology PURIFICATION AND KINETIC CHARACTERIZATION OF PROTOCHLOROPHYLLIDE a DIVINYL REDUCTASE IN GREEN SULFUR BACTERIUM CHLOROBIUM TEPIDUM A Thesis in Plant Physiology by Fang Shen © 2008 Fang Shen Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2008 The thesis of Fang Shen was reviewed and approved* by the following: Donald A. Bryant Ernest C. Pollard Professor of Biotechnology Professor of Biochemistry and Molecular Biology Thesis Advisor Ming Tien Professor of Biochemistry and Molecular Biology Teh-hui Kao Professor of Biochemistry and Molecular Biology Head of the Intercollege Graduate Program in Plant Biology *Signatures are on file in the Graduate School iii ABSTRACT (Bacterio)cholorophylls are magnesium tetrapyrroles involved in the light harvesting and photochemistry. In this thesis study, the bciA gene, encoding protochlorophyllide a divinyl reductase from Chlorobium tepidum, was overexpressed in Escherichia coli and purified by metal affinity chromatography. As estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) analysis, purified BciA was greater than 95% pure. Peptide mass fingerprinting of the overproduced BciA excised from a SDS-PAGE gel slice matched the CT1063 protein with a high score (202) and high coverage (70%). Non- denaturing PAGE showed that BciA mostly exist as monomers and that about 10% of the protein exists as dimers. Divinyl reductase assays using purified BciA confirmed that BciA was able to convert divinyl protochlorophyllide (DV-PChlide) into monovinyl protochlorophyllide (MV-PChlide) with NADPH serving as the reductant. NADH was not able to substitute for NADPH. Initial velocity studies suggest that BciA reacts via a ternary complex mechanism. Product inhibition studies indicated the binding sequence of the reactants. + NADPH binds first and NADP is released last. The Km value for the NADPH was calculated to be 20.1 ± 7.8 µM. The Km value for the DV-PChlide was estimated to be 1.7 ± 0.5 µM. These Km values are comparable to those of other enzymes involved in the (B)Chl biosynthesis pathways. The low Km values for tetrapyrroles are consistent with the fact that the tetrapyrroles are very toxic to the cells, and therefore the cellular concentrations of these compounds are most likely to be very low. iv TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................vii LIST OF TABLES.......................................................................................................ix LIST OF ABBREVIATIONS......................................................................................x ACKNOWLEDGEMENTS.........................................................................................xii Chapter 1 Introduction 1.1 Green sulfur Bacteria ..........................................................................................1 1.1.1 Ecology .....................................................................................................1 1.1.2 Phylogeny .................................................................................................2 1.1.3 Physiology ................................................................................................3 1.1.4 Photosynthetic apparatus of green sulfur bacteria ....................................4 1.1.5 Chlorosome...............................................................................................4 1.1.6 Reaction center..........................................................................................7 1.1.7 Energy transfer pathway ...........................................................................9 1.1.8 Chlorobium tepidum .................................................................................10 1.2 Pigment content and biosynthesis........................................................................11 1.2.1 Carotenoids in GSB ..................................................................................11 1.2.2 Structures of (bacterio)chlorophylls .........................................................12 1.2.3 Proposed model for (B)Chl biosynthesis in GSB.....................................13 1.2.4 Proposed model for BChl c biosynthesis in Chlorobium tepidum ...........14 1.2.5 Reduction of the C-8 vinyl group .............................................................15 1.3 The objectives of this thesis study .......................................................................16 v 1.4 References............................................................................................................17 Chapter 2 Purification and kinetic characterization of protochlorophyllide a divinyl reductase in C. tepidum 2.1 Introduction..........................................................................................................37 2.2 Materials and Methods 2.2.1 Cloning of the bciA gene and construction of the expression plasmid......37 2.2.2 Overproduction of BciA in E. coli.............................................................46 2.2.3 Purification of BciA from E. coli...............................................................47 2.2.4 Polyacrylamide gel electrophoresis ...........................................................48 2.2.5 Peptide mass fingerprinting ......................................................................48 2.2.6 Purification of divinyl protochlorophyllide ..............................................49 2.2.6.1 Culture of bchJ mutant of Chlorobium tepidum...................................49 2.2.6.2 HPLC purification of bchJ mutant spent medium ................................50 2.2.7 UV-Vis Spectroscopy ................................................................................51 2.2.8 BciA divinyl reductase activity assay........................................................51 2.2.9 Kinetic characterization of BciA as a DVR...............................................52 2.3 Results 2.3.1 Expression and purification of BciA.................................................................53 2.3.2 Purification of DV-and MV-PChlide................................................................54 2.3.3 Temperature dependence of specific activity of BciA......................................54 2.3.4 pH dependence of specific activity of BciA .....................................................55 2.3.5 DVR activity of purified BciA confirmed by HPLC........................................55 2.3.6 Initial velocity analysis ....................................................................................55 vi 2.3.7 Product inhibition analysis................................................................................56 2.4 Summary and Discussion.....................................................................................57 2.5 References............................................................................................................60 vii LIST OF FIGURES Figure 1.1 Phylogeny of the major eubacterial taxa based on RecA sequences........25 Figure 1.2 Phylogeny of green sulfur bacteria...........................................................26 Figure 1.3 The reductive tricarboxylic acid cycle for autotrophic CO2 fixation .......27 Figure 1.4 Structural organization of the photosystem I reaction center from Synechococcus elongates, and a representation of the equivalent complex from Chlorobium limicola ..................................................................................................28 Figure 1.5 Biosynthetic pathway of carotenoids in C. tepidum.................................29 Figure 1.6 Structure of bacterial chlorophylls ...........................................................30 Figure 1.7 Structure of Chl aPD .................................................................................31 Figure 1.8 Structure of BChl aPD ..............................................................................32 Figure 1.9 Structure of BChl cF ................................................................................33 Figure 1.10 Proposed pathway for BChl c biosynthesis in C. tepidum .....................34 Figure 1.11 Model for biosynthesis of (B)Chls from a central Chlide a intermediate................................................................................................................35 Figure 1.12 Reduction of DV-PChlide to MV-PChlide by a divinyl protochlorophyllide reductase (DVR) in C. tepidum.................................................36 Figure 2.1 Overproduction and purification of recombinant BciA............................70 Figure 2.2 Peptide mass fingerprinting identified BciA as the expression product of the gene CT1063....................................................................................................71 Figure 2.3 Non-denaturing polyacrylamide gel electrophoresis................................72 Figure 2.4 HPLC separation of DV-PChlide and MV-PChlide from bchJ spent medium ......................................................................................................................73 Figure 2.5 The absorption spectrum of DV-and MV-PChlide ..................................74 Figure 2.6 BciA confirmed by HPLC........................................................................76 viii Figure 2.7 Temperature dependence of the specific activity of BciA .......................76
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