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Purification and Characterization of Rhodobacter Sphaeroides Polyhistidine-Tagged Hema and Comparison with Purified Polyhistidine- Tagged Hemt

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Purification and Characterization of Rhodobacter Sphaeroides Polyhistidine-Tagged Hema and Comparison with Purified Polyhistidine- Tagged Hemt PURIFICATION AND CHARACTERIZATION OF RHODOBACTER SPHAEROIDES POLYHISTIDINE-TAGGED HEMA AND COMPARISON WITH PURIFIED POLYHISTIDINE- TAGGED HEMT Xiao Xiao A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2013 Committee: Dr. Jill Zeilstra-Ryalls, Ph.D., Advisor Dr. Rogers O. Scott Dr. Zhaohui Xu ii © 2013 Xiao Xiao All Rights Reserved iii ABSTRACT Jill Zeilstra-Ryalls, Ph.D, Advisor All tetrapyrrole, molecules that include heme, bacteriochlorophyll, and vitamin B12, are derived from 5-aminolevulinic acid (ALA). In the purple non-sulfur alphaproteobacteria Rhodobacter sphaeroides ALA is formed by the condensation of glycine and succinyl-CoA, catalyzed by the pyridoxal-phosphate dependent enzyme ALA synthase. Two ALA synthase genes, hemA and hemT are present in R. sphaeroides wild type strain 2.4.1. When expressed, either one of the gene products can satisfy the ALA requirement of the cell. Towards understanding the presence of two ALA synthases in one organism, each enzyme should be characterized individually in order to define what is similar and different about the enzymes. Using this information, one may be able to infer how the activities of the two ALA synthases are coordinate in R. sphaeroides. In this study, R. sphaeroides 2.4.1 recombinant polyhistidine- tagged HemA (rHemA) was affinity purified and its optimum temperature and pH, specific activity, and kinetic properties were determined. The effect of added hemin on its activity was also evaluated, as was its secondary structure composition using circular dichroism. These characteristics were then compared to those of recombinant polyhistidine-tagged HemT (rHemT). Two major differences were noted. First, the catalytic capacity of rHemA is more than ten times greater than rHemT. Second, rHemA has a higher affinity for succinyl-CoA than rHemT. A hypothesis that could explain the significance of these differences posits that HemT is needed to reduce ALA synthesis under conditions in which succinyl-CoA and glycine are in greater demand for metabolisms other than ALA formation, such as energy generation for the former and protein synthesis for the latter. Examining hemA and hemT expression under conditions that would impose such metabolic priorities would be appropriate to test this hypothesis. iv ACKNOWLEDGEMENTS Foremost, I would like to express my sincere appreciation to my advisor, Dr. Jill Zeilstra- Ryalls, for the opportunity to work in her laboratory. She offered huge support on my two years master program. I learn extensive research experience and became thinking critically because of her excellent guidance. Besides, I would like to thank the rest of my thesis committee, Dr. Scott O. Rogers and Dr. Zhaohui Xu for their warm encouragement and insightful comments. I would also thank Dr. Maneewan Suwansaard and my workmate James for their help with my experiments. v TABLE OF CONTENTS Page CHAPTER I: BACKGROUND INFORMATION AND SPECIFIC AIMS ……………….. 1 Introduction……………………………………………………………………………... 1 A. Rhodobacter sphaeroides ………………………………………………………… 1 B. Tetrapyrroles………………………………………………………………….……. 1 C. 5-Aminolevulinic acid (ALA) synthesis pathway…………………………..…….... 2 D. ALA synthases and its isozymes ………………………………………..…………. 4 Specific Aims…………………………………………………………………................. 5 CHAPTER II: PURIFICATION AND CHARACTERIZATION OF POLYHISTIDINE- TAGGED HEMA …………………………………………………………………………….. 7 Introduction………………………………………….………………………………......... 7 Materials and Methods………………………………………………………………….... 7 Bacterial strains, plasmids, and growth conditions………………………………….. 7 Crude lysate preparation and rHemA affinity purification……………………………. 9 Protein concentration determinations……………………………...…………………. 10 ALA synthase activity assays……………………………….………………………... 11 Determining pH and temperature optima of purified rHemA………….…………….. 12 Hemin addition analysis………………………………………..……………………… 12 Determination of Km, kcat, Vmax values for succinyl-CoA and glycine of rHemA……. 12 SDS-PAGE, immunoblot analysis, and InvisionTM in-gel stain………….............…. 13 Circular dichroism spectrometry and analysis……………………………………… 14 EKMaxTM treatment of rHemA…………………………………………….……..... 14 vi Protein modeling …………………………………………………………………….. 15 Results………………………………………………….…………................................... 15 Expression and purification of rHemA……………………………….…………..…... 15 Immunoblot analysis and in-gel polyhistidine staining of purified rHemA…...……… 18 Temperature and pH optima of purified rHemA…………………………….……….. 19 The effect of hemin on the ALA synthase activity of rHemA……………………… 20 Glycine and succinyl-CoA Km determinations for rHemA…………………………… 21 Circular dichroism (CD) analysis of rHemA.……………………………………...…. 22 Discussion……………………………………..…………………………………….......... 23 CHAPTER III: A COMPARISON OF THE PROPERTIES OF POLYHISTIDINE-TAGGED HEMA TO THOSE OF POLYHISTIDINE-TAGGED HEMT…………………..………….. 25 Introduction………………………………………….………………………………..... 25 Differences and similarities between purified rHemA and rHemT………………......... 26 REFERENCES……………………………………………………………………………….. 30 vii LIST OF FIGURES FIGURES Page 1 Schematic diagram of tetrapyrrole synthesis in R. sphaeroides. ……………………... 2 2 The two biosynthesis pathways by which ALA is formed. ………………………….. 3 3 Schematic diagram (A) of the plasmid vector pIND-hemA and amino acid sequence (B) of the product……………………………………………………….……………… 9 4 SDS-PAGE of the soluble and insoluble fractions of samples.………………………. 16 5 SDS-PAGE of samples collected during affinity purification of rHemA……………. 17 6 The amount of ALA formed per mg of protein versus assay incubation time……….. 18 TM 7 Immunoblot, and InVision in-gel staining of rHemA…………………………...… 19 8 Effect of temperature and pH on rHemA activity……...…………………………… 20 9 Effect of hemin on rHemA activity. …………………………………………………. 20 10 ALA synthase activity versus substrate concentrations, and nonlinear regression plots of the data used to determine glycine and succinyl-CoA Kms …………………. 21 11 Preliminary circular dichroic spectrum of purified rHemA.……………………….… 22 12 Amino acid sequence alignment of R. sphaeroides 2.4.1 wild type HemA and HemT generated using ClustalW ………………………………………………………........ 25 13 Tertiary structure model superpositions of R. sphaeroides 2.4.1 rHemA and wild type HemA upon the solved crystal structure of Rhodobacter capsulatus ALA………….. 26 viii LIST OF TABLES TABLES Page 1 Purification profile of rHemA………………………..……………………………… 17 2 Kinetic parameters for rHemA .………………………………………………..….. 22 3 Predicted, calculated, or known secondary structure compositions of R. sphaeroides rHemA, wild type HemA, and Rhodobacter capsulatus ALA synthase……………. 23 4 Comparison of the kinetic properties of rHemA and rHemT…….………………….. 27 ix LIST OF ABBREVIATIONS ALA 5-aminolevulinic acid ALAS 5-aminolevulinic acid synthase ATP adenosine triphosphate CD circular dichroism Cys cysteine Da dalton HCl hydrochloric acid IPTG Isopropyl β-D-1-thiogalactopyranoside kDa kilodalton Kn kanamycin LB Luria-Bertani M molar ml milliliter mM millimolar min minute nm nanometer OD optical density PLP pyridoxal 5’-phosphate psi pounds per square inch rHemA recombinant polyhistidine-tagged HemA rHemT recombinant polyhistidine-tagged HemT SDS sodium dodecyl sulfate x TCA trichloroacetic acid Trp tryptophan Tyr tyrosine β-ME β-mercaptoethanol ε molar extinction coefficient µg micrograms µl microliter 1 CHAPTER I: BACKGROUND INFORMATION AND SPECIFIC AIMS INTRODUCTION A – Rhodobacter sphaeroides R. sphaeroides belongs to the Class alpha-proteobacteria which has been isolated from both fresh and salt water throughout the world (1, 2). These remarkably metabolically versatile bacteria are capable of hydrogen production (3), atmospheric nitrogen and carbon dioxide fixation, and can obtain energy through aerobic respiration, anaerobic respiration or anoxygenic photosynthesis (reviewed in ref. 4). Because of their versatility they have served as model organisms in both applied and basic research. Such studies include exploring ways to exploit their useful metabolic capabilities and investigating how such wide-ranging metabolisms are efficiently orchestrated. B – Tetrapyrroles Tetrapyrroles, are composed of four pyrrole rings that are connected with each other by a methane or methylene unit. Rhodobacter sphaeroides is capable of synthesizing three kinds of biologically relevant cyclic tetrapyrroles, heme, bacteriochlorophyll, and vitamin B12, via a branching biosynthetic pathway (reviewed in ref. 5; Fig. 1). These molecules play vital roles in various catabolic and anabolic biological processes in bacteria. Electron transferring hemes are contained in many proteins like cytochromes that are involved in both aerobic and photosynthetic electron transport chains (reviewed in ref. 6). Bacteriochlorophyll is responsible for light-harvesting in the photosynthesis system of R. sphaeroides. Vitamin B12 is an essential cofactor for many important anabolic enzymes, such as methionine synthase (reviewed in ref. 7). As could be predicted from their roles in these bacteria, the production of these different 2 tetrapyrroles is tightly regulated and their relative and absolute amounts change significantly, according to environmental conditions. For example, when light is present under anaerobic conditions, R. sphaeroides synthesizes a large amount of bacteriochlorophyll to support anoxygenic photosynthesis, while under aerobic conditions bacteriochlorophyll synthesis blocked because
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