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Methanocaldococcus Jannaschii and the Recycling of S-Adenosyl-L-Methionine (SAM)

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Methanocaldococcus Jannaschii and the Recycling of S-Adenosyl-L-Methionine (SAM) Methanocaldococcus jannaschii and the Recycling of S-Adenosyl-L-methionine (SAM) Danielle V. Miller Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biochemistry Robert H. White, Chair Pablo Sobrado Timothy J. Larson David R. Bevan March 16th, 2017 Blacksburg, VA Keywords: S-adenosyl-L-methionine, SAM, recycling, 6-deoxy-5-ketofructose 1- phosphate, aromatic amino acids, methionine salvage, 5’-deoxyadenosine, S- adenosylhomoysteine, methylthioadenosine Methanocaldococcus jannaschii and the recycling of S-Adenosyl-L-methionine (SAM) Danielle V. Miller ABSTRACT S-Adenosyl-L-methionine (SAM) is an essential metabolite for all domains of life. SAM- dependent reactions result in three major metabolites: S-adenosyl-L-homocysteine (SAH), methylthioadenosine (MTA), and 5’-deoxyadenosine (5’-dA). Each of these has been demonstrated to be feedback inhibitors of SAM dependent enzymes. Thus, each metabolite has a pathway to prevent inhibition through the salvage of nucleoside and ribose moieties. However, these salvage pathways are not universally conserved. In the anaerobic archaeal organism Methanocaldococcus jannaschii, the salvage of SAH, MTA, and 5’-dA, proceeds first via deamination to S-inosylhomocysteine (SIH), methylthioinosine (MTI), and 5’-deoxyinosine (5’-dI). The annotated SAH hydrolase from M. jannaschii is specific for SIH and the hydrolyzed product homocysteine is then methylated to methionine. The salvage of MTA is known to proceed through the methionine salvage pathway, however, an anaerobic route for the salvage of MTA is still mostly unknown. Only two enzymes from the methionine salvage pathway are annotated in M. jannaschii’s proteome, a methylthioinosine phosphorylase (MTIP) and methylthioribose 1-phosphate isomerase (MTRI). These enzymes were shown to produce methylthioribulose 1-phosphate from MTI. Unfortunately, how MTI is converted to either 2-keto-(4-methylthio)butyrate or methionine remains unknown. The two enzymes involved in the salvage of MTI have also been demonstrated to be involved in the salvage of 5’-dI. Interestingly, there is little information on how 5’-dA or 5’-dI is recycled and it is proposed here to be the source of deoxysugars for the production methylglyoxal, a precursor for aromatic amino acids. MTIP and MTRI were demonstrated to produce 5- deoxyribulose 1-phosphate from 5’-dI. Additionally, two enzymes annotated as part of the pentose phosphate pathway, ribulose 5-phosphate 3-epimerase and transketolase, were able to convert 5-deoxyribulose 1-phosphate to lactaldehyde. Lactaldehyde was then reduced to methylglyoxal by an essential enzyme in methanogenesis, N5, N10- methylenetetahydromethanopterin reductase with NADPH. These results further demonstrate a novel route for the biosynthesis of methylglyoxal. Lastly, hypoxanthine produced from phosphorolysis of inosine, MTI, and 5’-dI was demonstrated to be reincorporated through the hypoxanthine/guanine phosphoribosyltransferase (Hpt) to IMP. Together these reactions represent novel pathways for the salvage of the SAM nucleoside and ribose moieties in M. jannaschii. GENERAL AUDIENCE ABSTRACT In the anaerobic methanogenic archaea Methanocaldococcus jannaschii traditional metabolic pathways are often missing or incomplete and are substituted by unique ones. M. jannaschii is deeply rooted on the phylogenetic tree and serves as a model organism for the study of primitive metabolism. Discussed here are the recycling pathways of the essential cofactor S-adenosyl-L-methionine (SAM). SAM recycling pathways in Archaea have not been investigated prior to this work. Two of the universal pathways responsible for recycling SAM to methionine were found to be modified and unique. A third pathway was proposed that would be responsible for generating an essential precursor for the biosynthesis of aromatic amino acids. The identification of the pathways and enzymes from M. jannaschii will give insight into the biochemical reactions that were occurring when life originated. Eight enzymes are discussed here that demonstrate how the recycling pathways in M. jannaschii are interconnected and the enzymes are shared between them. This work further describes the importance of understanding these unique microorganisms and the metabolic pathways they utilize to help understand primitive life. Acknowledgments I would like to thank Dr. Robert H. White for the time and effort he has put into the development of my scientific abilities. He has been instrumental in shaping the scientist I have become and will continue to be. He is always challenging the norm, which has opened my mind about the type of reactions that enzymes and nature are capable of performing. His passion and enthusiasm for science and research has left a lasting impression on me that I hope to pass on to my own students one day. I would additionally like to thank my committee members, Dr. Bevan, Dr. Sobrado, and Dr. Larson for challenging me and always being supportive of my career objectives. I also want to thank everyone in the department of Biochemistry for all their help and support over the years. Lastly, I would like to thank my friends and family for their support and encouragement these past five years of graduate school. iv Table of Contents Introduction ....................................................................................................................... 1 Tables ............................................................................................................................. 9 Table 1 ........................................................................................................................ 9 Table 2 ........................................................................................................................ 9 Figures .......................................................................................................................... 10 Figure 1 ..................................................................................................................... 10 Figure 2 ..................................................................................................................... 11 Figure 3 ..................................................................................................................... 12 Figure 4 ..................................................................................................................... 13 Figure 5 ..................................................................................................................... 14 Figure 6 ..................................................................................................................... 15 Chapter 1: S-Inosylhomocysteine Hydrolase: A novel enzyme involved in SAM recycling ............................................................................................................................ 16 Tables ........................................................................................................................... 34 Table 1 ...................................................................................................................... 34 Table 2 ...................................................................................................................... 34 Figures .......................................................................................................................... 36 Figure 1 ..................................................................................................................... 36 Figure 2 ..................................................................................................................... 37 Figure 3 ..................................................................................................................... 39 Figure 4 ..................................................................................................................... 40 Chapter 2: Promiscuity of Methionine Salvage Pathway Enzymes in Methanocaldococcus jannaschii and Methanosarcina acetivorans ................................. 41 Table 1 ...................................................................................................................... 65 Table 2 ...................................................................................................................... 65 Figures .......................................................................................................................... 66 Figure 1 ..................................................................................................................... 66 Figure 2 ..................................................................................................................... 67 Figure 3 ..................................................................................................................... 68 Figure 4 ..................................................................................................................... 69 Figure 5 ..................................................................................................................... 70 Supporting Information ............................................................................................. 71 Table S1 .................................................................................................................... 72 Table S2 ...................................................................................................................
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