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. / Ph. D. / In the anaerobic methanogenic archaea <i>Methanocaldococcus jannaschii</i> traditional metabolic pathways are often missing or incomplete and are substituted by unique ones. <i>M. jannaschii</i> 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 <i>Archaea</i> 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 <i>M. jannaschii</i> 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 <i>M. jannaschii</i> 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.

Strikt anaerobe Bakterien wie Clostridium difficile und C. scatologenes verwenden GRE, um die chemisch ungünstige Decarboxylierung von 4-Hydroxyphenylacetat zu p-Cresol zu katalysieren. Das Enzymsystem besteht aus einer Decarboxylase und dem zugehörigen Aktivierungsenzym. Die 4-Hydroxyphenylacetat-Decarboxylase (4Hpad) besitzt zusätzlich zum Protein-basierten Glycinradikal eine weitere Untereinheit mit bis zu zwei [4Fe-4S] Clustern und repräsentiert hierdurch eine neue Klasse von Fe/S-Cluster-haltigen GREs, die aromatische Verbindungen umsetzen. Das Aktivierungsenzym (4Hpad-AE) weicht vom Standardtypus ab, indem es zusätzlich zum S-Adenosylmethionin(SAM)-bindenden [4Fe-4S]-Cluster (RS-Cluster) mindestens einen weiteren [4Fe-4S]-Cluster bindet. In dieser Studie wurden heterologe Expressions- und Reinigungsprotokolle für 4Hpad und 4Hpad-AE entwickelt. Kristallstrukturen von 4Hpad cokristallisiert mit den Substraten (4-Hydroxyphenylacetat, 3,4-Dihydroxyphenylacetat) und dem Inhibitor (4-Hydroxyphenylacetamid) zeigten geringe strukturelle Änderungen im aktiven Zentrum des Proteins. Die Radikalbildung am 4Hpad-AE wurde durch die Überprüfung einer klassischen reduktiven Spaltung von SAM zu den Reaktionsprodukten 5’-Deoxyadenosin und Methionin bestätigt. EPR- und Mössbauer-Spektroskopische Analysen zeigten, dass 4Hpad-AE mindestens einen zusätzlichen [4Fe-4S] Cluster neben dem einzelnen RS-Cluster enthält. Die katalytische Notwendigkeit eines zusätzlichen Clusters wurde durch eine Mutationsanalyse untersucht, wobei eine verkürzte Version des Enzyms ohne die zusätzliche Cystein-reiche Insertion konstruiert wurde. Das verkürzte Mutante ohne die Bindungsmotive für die zusätzlichen Cluster gekennzeichnet, die Konfiguration, Stöchiometrie und die Funktion der zusätzlichen Cluster diagnostizieren. / 4-hydroxyphenylacetate decarboxylase (4Hpad) is a two [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in Clostridium difficile and C. scatologenes by a Kolbe-type decarboxylation. The decarboxylation product p-cresol is a virulence factor of the human pathogen C. difficile. The small subunit of 4Hpad may have a regulatory function with the Fe/S clusters involved in complex formation and radical dissipation in the absence of substrate. The respective activating enzyme (4Hpad-AE) has one or two [4Fe-4S] cluster(s) in addition to the SAM-binding [4Fe-4S] cluster (RS cluster). The role of these auxiliary clusters is still under debate with proposed functions including structural integrity and conduit for electron transfer to the RS cluster. This study shows the optimized expression and purification protocols for the decarboxylase and the co-crystallization experiments and binding studies with 4-hydroxy-phenylacetate and 3,4-dihydroxyphenylacetate and with the inhibitor 4-hydroxy-phenylacetamide. The purification and characterization of active site mutants of decarboxylase are also done. Concerning 4-HPAD-AE, we report on the purification of code-optimized variants, and on spectroscopic and kinetic studies to characterize the respective i) SAM binding enthalpies, ii) rates for reductive cleavage of SAM and iii) putative functions of the additional Fe/S clusters. The truncated mutant lacking the binding motifs for the auxiliary clusters is characterized to diagnose the configuration, stoichiometry and function of the auxiliary clusters.