Evolutionary History of Nickel-Dependent Enzymes: Implications for the Origins of Life. Reem Hallak Degree project in biology, Master of science (2 years), 2020 Examensarbete i biologi 60 hp till masterexamen, 2020 Biology Education Centre and Department of Earth Sciences, Palaeobiology, Uppsala University Supervisors: Aodhán Butler and Anna Neubeck External opponent: Brenda Irene Medina Jimenez Table of Contents ABSTRACT .................................................................................................................................................. 5 INTRODUCTION ...................................................................................................................................... 6 METHODOLOGY ................................................................................................................................... 11 ▪ Brief Summary .................................................................................................................................................... 11 ▪ Operating Principle of HMMER ................................................................................................................. 11 ▪ Detailed Summary - Collecting Query Sequences ............................................................................ 13 a. From Published Study: ............................................................................................................................... 13 b. From Databases:........................................................................................................................................... 13 ▪ Searching for Homologs ................................................................................................................................ 14 a. Concatenating the Sequences: ............................................................................................................... 14 b. Setting Up the Sequence Databases: .................................................................................................. 14 c. Building the Profile HMMs + Conducting the Search:................................................................. 15 d. Visualizing the Results: ............................................................................................................................... 16 ▪ Gene Trees ......................................................................................................................................................... 16 a. Building the Trees: ....................................................................................................................................... 16 b. Annotation & Visualization of the Trees:........................................................................................... 16 c. Comparison of Tree Topologies: ......................................................................................................... 17 ▪ Ancestral State Reconstruction .................................................................................................................. 17 RESULTS .................................................................................................................................................... 18 ▪ HMMER Results Summary ............................................................................................................................ 18 Page 1 of 68 ▪ Gene Trees + Ancestral State Reconstruction ................................................................................... 18 A. Redox Enzymes ................................................................................................................................................. 18 1. Co-methylating acetyl-CoA synthase (Enzymatic complex of ACS & CODH)............... 18 2. Carbon monoxide dehydrogenase (Ni-CODH) ........................................................................... 20 3. Methyl-coenzyme M reductase (MCR) .............................................................................................. 22 4. Superoxide dismutase (Ni-SOD) ......................................................................................................... 23 5. Coenzyme F420 hydrogenase (FrhABG): A Group 3a Ni-Fe hydrogenase ......................... 25 6. Hydrogen:quinone oxidoreductase (or quinone-reactive Ni-Fe hydrogenase (Group 1)) 26 7. Ni-dependent hydrogenase (NiFeSe_Hases) .................................................................................. 28 B. Non-redox Enzymes ....................................................................................................................................... 30 1. Acireductone dioxygenase (Ni-ARD) ................................................................................................ 30 2. Glyoxalase I (GloI or GlxI) ....................................................................................................................... 32 3. Lactate racemase (LarA) .......................................................................................................................... 34 4. Urease............................................................................................................................................................... 35 ▪ Tree Comparison ............................................................................................................................................. 37 → CO-methylating acetyl-CoA synthase ........................................................................................... 38 → Carbon monoxide dehydrogenase (Ni-CODH) ...................................................................... 38 ▪ Supplementary Files ......................................................................................................................................... 38 DISCUSSION ............................................................................................................................................ 38 ▪ General Trends in Ancestral State Reconstruction Results ........................................................... 38 ▪ The Analogous Histories within Nickel Enzyme Groups: Single or Multiple Origins? ........ 39 Page 2 of 68 ▪ Selective Pressures: Nickel Famine or the Great Oxidation Event? ........................................... 40 ▪ The Odd Case of Methyl-coenzyme M Reductase ...................................................................... 41 ▪ Partial Genes in the LCA of Prokaryotes .......................................................................................... 42 ▪ Non-redox Enzymes: Convergent Evolution or LGT? ..................................................................... 42 ▪ Conflicting Tree Topologies: Evidence of Lateral Gene Transfer (LGT)? ................................ 43 ▪ Tree Comparison Results ........................................................................................................................ 45 → CO-methylating acetyl-CoA synthase ........................................................................................... 45 → Carbon monoxide dehydrogenase (Ni-CODH) ...................................................................... 48 ▪ Implications for the Origin of Life .............................................................................................................. 50 → Why are nickel enzymes interesting for the origins of life?................................................... 50 → Implications from this study. ............................................................................................................... 51 → In what context do these new results matter? .......................................................................... 52 ▪ Inherent Assumptions of the Models....................................................................................................... 54 CONCLUSION ........................................................................................................................................ 55 ACKNOWLEDGEMENTS ..................................................................................................................... 56 REFERENCES ............................................................................................................................................ 58 LIST OF APPENDICES ........................................................................................................................... 67 Appendix A: Lophotrochozoa Hits .................................................................................................................... 67 Appendix B: Lingula reevi Hits ............................................................................................................................. 67 Appendix C: Occupancy Matrices ...................................................................................................................... 67 Appendix D: Gene Trees ....................................................................................................................................... 67 Appendix E: Ancestral State Reconstruction (ASR) Trees ...................................................................... 67 Page 3 of 68 Appendix F: Tanglegrams ....................................................................................................................................... 67 Appendix G: Reference Tree...............................................................................................................................