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Examination of Mutants That Alter Oxygen Sensitivity and Co2/O2 Substrate Specificity of the Ribulose 1,5-Bisphosphate Carboxyla

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Examination of Mutants That Alter Oxygen Sensitivity and Co2/O2 Substrate Specificity of the Ribulose 1,5-Bisphosphate Carboxyla EXAMINATION OF MUTANTS THAT ALTER OXYGEN SENSITIVITY AND CO2/O2 SUBSTRATE SPECIFICITY OF THE RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) FROM ARCHAEOGLOBUS FULGIDUS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nathaniel E. Kreel, B.S. ***** The Ohio State University 2008 Dissertation Committee: Professor Dr. F. Robert Tabita, Advisor Approved by Professor Dr. Charles E. Bell Professor Dr. Charles L. Brooks Professor Dr. Michael Ibba ____________________________ Advisor Ohio State Biochemistry Graduate Program ABSTRACT The archaeon Archaeoglobus fulgidus contains a gene (rbcL2) that encodes the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme necessary for biological reduction and assimilation of CO2 to organic carbon. Based on sequence homologies and phylogenetic differences, archaeal Rubiscos represent a special class of Rubisco, termed form III, that distinguishes it from the previously characterized form I and form II enzymes. Form III Rubisco retains many features characteristic of all forms of Rubisco, yet exhibits many interesting and unique differences that might be exploited to learn more about structure-function relationships for this protein. For example, recombinant A. fulgidus RbcL2 was shown to possess an extremely high kcat value (23 s-1) and optimal activity was reached at temperatures up to 93°C. Furthermore, this protein was unusual in that exposure or assay in the presence of O2 (in the presence of high levels of CO2) resulted in substantial loss (90%) in activity compared to assays performed under strictly anaerobic conditions. Kinetic studies indicated that A. fulgidus RbcL2 possessed an unusually high affinity for O2. Comparative bioinformatic analyses of available archaeal Rubisco sequences suggested the potential importance of several unique residues, as did further analyses within the context of available forms I/II/III Rubisco structures. Two residues unique to archaeal Rubisco, Met-295 and Ser-363, were of particular interest due to their proximity to known active site residues. ii Moreover, it was shown that recombinant mutant M295D, S363I and S363V A. fulgidus enzymes were less sensitive to oxygen compared to the wild-type protein. The unique oxygen sensitivity of this form III archaeal Rubisco is being used as a model to provide clues as to how Rubisco has evolved to become more stable in the presence of oxygen in more evolutionarily advanced form I and form II proteins. In addition, the same mutational changes were made at identical sites in Thermococcus kodakaraensis RbcL which shares a 72% amino acid sequence identity with A. fulgidus RbcL2. Although this Rubisco enzyme from T. kodakaraensis is not as oxygen sensitive as the Rubisco from A. fulgidus, there are many similarities in oxygen sensitivity not observed in other form I and form II Rubiscos. A. fulgidus RbcL2 is able to complement growth in the double Rubisco knockout strain SBI/II- from Rhodobacter capsulatus. For the first time, using the A. fulgidus RbcL2 enzyme to complement growth in this knockout strain has enabled the wild-type and mutant enzymes to be studied in vivo. iii Dedicated to all who have traveled along in this journey with me, through the ups and downs, the good times and the not so good times, there is no way I could have made it without your love and support. No names need to be mentioned, you know who you are. iv ACKNOWLEDGMENTS I would like to thank my advisor, F. Robert Tabita, for creating a working environment where several different avenues of research could be investigated. I thank Dr. Stephanie Scott and Dr. Cedric Bobst for their consistent technical assistance throughout my thesis. I am grateful to my coworkers, both past and present, for giving me scientific and computer advice. This research was supported by a grant from the National Institute of Health. v VITA May 6, 1978 ................................................Born – Fayetteville, NC 2000.............................................................B.S. Biology, East Carolina University 2000-2001 ...................................................Biochemistry Research Technician Novartis Biotechnology 2001 – present.............................................Graduate Research Associate, The Ohio State University PUBLICATIONS Research Publications 1. Kreel, N.E. and Tabita, F.R. 2007. Substitutions at Methionine 295 of the Archaeoglobus fulgidus ribulose-1,5-bisphosphate carboxylase/oxygenase affects oxygen binding and CO2/O2 specificity. J. Biol Chem. 282:1341- 51. 2. Tabita, F.R., Satagopan, S., Hanson, T.E., Kreel, N.E., and Scott, S.S. Distinct form I, II, III and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. J. Exp. Botany. In press 2008. 3. Tabita, F.R., Hanson, T.E., Satagopan, S., Witte, B.H., and Kreel, N.E. The evolution, structure and function of Rubisco and its homolog the Rubisco-like protein. Manuscript submitted. 2008. 4. Kreel, N.E. and Tabita, F.R. Activity and carboxylation specificity factor of mutants of ribulose 1,5-bisphosphate carboxylase/oxygenase from Archaeoglobus fulgidus. Manuscript submitted. 2008. FIELDS OF STUDY Major Field: Biochemistry vi TABLE OF CONTENTS Abstract......................................................................................................................ii Dedication..................................................................................................................iv Acknowledgments......................................................................................................v Vita.............................................................................................................................vi List of Tables .............................................................................................................ix List of Figures............................................................................................................x List of Abbreviations .................................................................................................xiv Chapters: 1. Introduction....................................................................................................1 2. Kinetic properties of Archaeoglobus fulgidus RbcL2; site-directed mutangenesis studies to probe the basis for oxygen sensitivity.....................21 Introduction....................................................................................................21 Materials and Methods...................................................................................26 Results............................................................................................................32 Discussion......................................................................................................59 3. Further studies of mutant ribulose 1,5-bisphosphate carboxylase/oxygenase proteins from Archaeoglobus fulgidus and a related archaeon......................66 Introduction....................................................................................................66 Materials and Methods...................................................................................70 Results............................................................................................................82 Discussion......................................................................................................118 vii 4. Summary........................................................................................................134 Future Experiments........................................................................................145 References..................................................................................................................148 viii LIST OF TABLES Table Page 2.1 Kinetic properties of purified, recombinant wild-type and mutant M295D Rubisco from A. fulgidus RbcL2..........................................51 3.1 Plasmids and bacterial strains ............................................................81 3.2 Amino acid comparison in hydrophobic pocket of form I, II and III Rubiscos........................................................................................83 3.3 Kinetic properties of purified wild-type and mutant enzymes from A. fulgidus RbcL2 ..............................................................................87 3.4 Carboxylase activity of heat stable extracts from A. fulgidus RbcL2 wild-type and mutant enzymes under anaerobic and oxygen exposed conditions.............................................................................94 3.5 Carboxylase activity of heat stable extracts from A. fulgidus RbcL2 wild-type and mutant enzymes under anaerobic and oxygen exposed conditions.............................................................................99 3.6 Carboxylase activity of crude soluble extracts of A. fulgidus RbcL2 wild-type and mutant enzymes from R. capsulatus SBI/II- grown under photoheterotrophic conditions ...........................................................107 3.7 Kinetic properties of purified wild-type Rubisco from T. kodakaraensis RbcL ..........................................................................116 3.8 Carboxylase activity of heat stable extracts from T. kodakaraensis RbcL wild-type and mutant enzymes under anaerobic and oxygen exposed conditions
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