Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme James Kaganjo 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: Jill Zeilstra-Ryalls, Ph.D, Advisor Raymond Larsen, Ph.D Scott Rogers. Ph.D © 2013 James Kaganjo All Rights Reserved iii ABSTRACT Jill Zeilstra-Ryalls, Advisor Rhodobacter sphaeroides synthesizes heme, vitamin B12 and bacteriochlorophyll to support its metabolic versatility that includes the ability to obtain energy by chemo- and phototrophy. The common precursor of all tetrapyrroles is 5-aminolevulinic acid (ALA) which in these bacteria is formed from the condensation of glycine and succinyl- CoA, a reaction catalyzed by PLP-dependent ALA synthase. ALA synthase is a member of the class II of fold type I PLP-dependent enzymes that also includes 2-amino-3- ketobutyrate-CoA ligase (KBL), serine palmitoyltransferase (SPT) and 8-amino-7- oxononanoate synthase (AONS). In R. sphaeroides strain 2.4.1, there are two ALA synthase genes, hemA and hemT, and when expressed each one alone can satisfy the requirement for ALA under all conditions that have been examined. In order to understand the role of the two ALA synthase isoenzymes, purification, characterization and comparison of the characteristics of both isoenzymes is necessary. In this study, recombinant polyhistidine-tagged HemT (rHemT) was purified using nickel affinity chromatography, and it was characterized in terms of optimum pH and temperature, the effect of added hemin, its kinetic properties, its secondary structure, the presence of disulfide bonds, and its activity using an alternative substrate. The properties of rHemT were then compared to those of recombinant polyhistidine-tagged HemA (rHemA), which was analyzed separately. The specific activity of rHemT was more than 10-fold lower than that of rHemA. The glycine Km for both proteins was the same, although the catalytic turnover rate for glycine was more than 10-fold higher for iv rHemA than for rHemT. The succinyl-CoA Km for rHemA was 2-fold lower than for rHemT, and again the catalytic turnover was 10-fold higher for rHemA than for rHemT. These differences indicate that HemT is a low activity ALA synthase when compared to HemA, which would be explained if HemT has a greater preference for substrates other than succinyl-CoA. Towards this end, the ability of rHemT to use acetyl-CoA was evaluated. Using purified rHemT no detectable product was formed, indicating that succinyl-CoA is the preferred substrate. Together with the comparisons of the HemA and HemT characteristics, a consideration of the substrates used in ALA production has suggested a possible explanation for the need to have both enzymes. This relies on the hypotheses that, (1) when hemA and hemT are both expressed, the products are able to form heterodimeric proteins, and (2) such heterodimers are less active than HemA homodimers, although possibly more active than HemT homodimers; i.e. hemT behaves in a dominant negative fashion to hemA. If true, then the role of HemT could be to reduce ALA synthase activity in the cell when succinyl-CoA and/or glycine are required for energy metabolism and protein synthesis, respectively. v ACKNOWLEDGMENTS I would like to thank Dr. Zeilstra-Ryalls for her immense guidance and mentorship for the last two years and also for giving me the opportunity to work in her laboratory. I would also like to express my gratitude to the members of my committee Dr. S. Rogers and Dr. R. Larsen for their understanding and support during my study. To Dr. M. Suwansaard (Joy), I am forever indebted for her guidance and mentorship when I joined the lab and throughout my master’s study. To all our lab members past and current am grateful for their helpful comments and ideas. Finally I would like to acknowledge my friends and family for their support and guidance. vi TABLE OF CONTENTS Page CHAPTER 1. BACKGROUND INFORMATION AND SPECIFIC AIMS ....................... 1 INTRODUCTION ..................................................................................................... 1 Rhodobacter sphaeroides............................................................................... 1 Tetrapyrroles .................................................................................................. 2 ALA biosynthesis........................................................................................... 4 ALA synthase isoenzymes ............................................................................. 7 SPECIFIC AIMS ....................................................................................................... 8 CHAPTER 2. PURIFICATION AND CHARACTERIZATION OF ACTIVE POLYHISTIDINE-TAGGED HemT ENZYME ................................................................... 10 INTRODUCTION … ................................................................................................ 10 MATERIALS AND METHODS ............................................................................... 10 Bacterial strains, plasmids and growth conditions ......................................... 10 rHemT affinity purification ............................................................................ 12 Determination of rHemT protein concentration ............................................ 13 ALA synthase and 2-amino-3-ketobutyrate CoA ligase (KBL) activity assays .................................................................................... 14 pH and temperature profile determinations ................................................... 15 Effect of added hemin .................................................................................... 15 Kinetic constant determinations ..................................................................... 15 Disulfide bond analysis .................................................................................. 16 Treatment with 4-acetamido-4’-maleimidylstilbene-2,2’- vii disulfonic acid .................................................................................... 16 Treatment with 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole ................ 16 Circular dichroism spectroscopy.................................................................... 17 SDS-PAGE .................................................................................................... 18 Immunoblot analysis ...................................................................................... 18 InVision™ His-tag In-gel Stain assay ........................................................... 19 Protein modeling and bioinformatic analysis ................................................ 19 Chemicals, reagents, and antisera .................................................................. 19 RESULTS ............................................................................................................ 20 Expression and purification of rHemT .......................................................... 20 SDS-PAGE, immunoblot analysis and InVisionTM His-tag analysis............. 21 Protein concentration determination .............................................................. 22 Optimum Temperature and pH ...................................................................... 23 Effect of added hemin .................................................................................... 23 Glycine and Succinyl-CoA Km determinations .............................................. 24 Is HemT an ALA synthase or a KBL? ........................................................... 25 Circular dichroism (CD) analysis of rHemT secondary structure ................. 27 Disulfide bond determination ........................................................................ 29 DISCUSSION ............................................................................................................ 31 CHAPTER 3. COMPARISON OF R. sphaeroides 2.4.1. POLYHISTIDINE-TAGGED HemA WITH POLYHISTIDINE-TAGGED HemT ......................................................................... 32 INTRODUCTION .................................................................................................... 32 PROPERTIES OF PURIFIED R. sphaeroides 2.4.1 rHemA versus rHemT ............ 32 viii DISCUSSION ........................................................................................................... 36 REFERENCES ...................................................................................................................... 38 ix LIST OF FIGURES/TABLES Figures Page 1 Diagram showing the basic structure of tetrapyrroles ............................................... 2 2 Schematic diagram of tetrapyrrole biosynthesis in Rhodobacter sphaeroides .......... 4 3 The two pathways of 5-aminolevulinic acid biosynthesis ......................................... 5 4 Schematic representation of the plasmid map of the expression vector pIND4-hemT 11 5 The amount of ALA formed per mg of protein versus assay incubation time .......... 21 6 Immunoblot and His-tag staining of rHemT. ............................................................. 22 7 Effect of pH and temperature on rHemT activity ...................................................... 23 8 Effect of added hemin on rHemT activity ................................................................. 24 9 Substrate concentration versus initial velocity (Vo) and non-linear regression plots for the determination