Methionine is essential for proper functioning of cellular processes such as protein synthesis, transmethylation and polyamine synthesis. Efficient recycling of methionine is important because of its limited bioavailability and metabolically expensive de novo synthesis. Further, cellular accretion of the nucleoside metabolites of the methionine salvage pathway compromises polyamine biosynthesis, transmethylation reactions and quorum sensing pathways, all critical reactions in cellular metabolism. 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a key component of the methionine salvage pathway of plants and many bacterial species, including Escherichia coli, Enterococcus faecalis, Salmonella typhimerium, Haemophilus influenza and Streptococcus pneumoniae. In bacteria, this enzyme displays dual-substrate specificity for two methionine metabolites, 5’-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), and catalyzes the irreversible cleavage of the glycosidic bond to form adenine and the corresponding thioribose products, methylthioribose (MTR) and S-ribosylhomocysteine (SRH), respectively. In plants, MTAN is highly specific towards MTA and shows 0-16 % activity towards SAH. Plants rely on SAH hydrolase to metabolize SAH. Mammals do not have the nucleosidase enzyme and MTA is metabolized by MTA phosphorylase (MTAP). Like plants, mammals utilize SAH hydrolase to degrade SAH. Because MTAN is required for viability in multiple bacterial species and is not found in humans, it has been identified as a target for novel antibiotic development. This thesis describes the structural and functional characterization of bacterial and plant MTANs, with the aim of better understanding the molecular determinants of substrate specificity and the catalytic mechanism of this enzyme. The catalytic activities of representative plant MTANs from Arabidopsis thaliana, AtMTAN1 and AtMTAN2, were kinetically characterized. While AtMTAN2 shows 14 % activity towards SAH relative to MTA, AtMTAN1 is completely inactive towards SAH. As such, AtMTAN1 was selected for further examination and comparison with the bacterial MTAN from Escherichia coli (EcMTAN). The structures, dynamics and thermodynamic properties of these enzymes were analyzed by X-ray crystallography, hydrogen-exchange coupled mass spectrometry and isothermal titration calorimetry, respectively. Our studies reveal that structural differences alone do not sufficiently explain the divergence in substrate specificity, and that conformational flexibility also plays an important role in substrate selection in MTANs. MTANs from the pathogenic bacterial species, Staphylococcus aureus and Streptococcus pneumoniae, were examined kinetically and structurally. Comparison of the structures and catalytic activities of these enzymes with EcMTAN shows that the discrepancies in kinetic activities arefully explained by structural differences, as the overall structure and active sites of these bacterial MTANs are nearly identical. These experiments are in agreement with our proposal that dynamics play a significant role in catalytic activity of MTAN, and suggest that both structure and dynamics must be considered in future antibiotic design. To further our understanding on the catalytic mechanism of MTAN, the putative catalytic residues of AtMTAN1 were identified by structural comparison to EcMTAN and mutated by site-directed mutagenesis. The AtMTAN1 mutants were analyzed by circular dichroism and kinetic studies. Our results suggest that the catalytic mechanism is largely conserved between bacterial and plant MTANs, although the role of the putative catalytic acid remains to be confirmed.

Methionine is essential for proper functioning of cellular processes such as protein synthesis, transmethylation and polyamine synthesis. Efficient recycling of methionine is important because of its limited bioavailability and metabolically expensive de novo synthesis. Further, cellular accretion of the nucleoside metabolites of the methionine salvage pathway compromises polyamine biosynthesis, transmethylation reactions and quorum sensing pathways, all critical reactions in cellular metabolism. 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a key component of the methionine salvage pathway of plants and many bacterial species, including Escherichia coli, Enterococcus faecalis, Salmonella typhimerium, Haemophilus influenza and Streptococcus pneumoniae. In bacteria, this enzyme displays dual-substrate specificity for two methionine metabolites, 5’-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), and catalyzes the irreversible cleavage of the glycosidic bond to form adenine and the corresponding thioribose products, methylthioribose (MTR) and S-ribosylhomocysteine (SRH), respectively. In plants, MTAN is highly specific towards MTA and shows 0-16 % activity towards SAH. Plants rely on SAH hydrolase to metabolize SAH. Mammals do not have the nucleosidase enzyme and MTA is metabolized by MTA phosphorylase (MTAP). Like plants, mammals utilize SAH hydrolase to degrade SAH. Because MTAN is required for viability in multiple bacterial species and is not found in humans, it has been identified as a target for novel antibiotic development. This thesis describes the structural and functional characterization of bacterial and plant MTANs, with the aim of better understanding the molecular determinants of substrate specificity and the catalytic mechanism of this enzyme. The catalytic activities of representative plant MTANs from Arabidopsis thaliana, AtMTAN1 and AtMTAN2, were kinetically characterized. While AtMTAN2 shows 14 % activity towards SAH relative to MTA, AtMTAN1 is completely inactive towards SAH. As such, AtMTAN1 was selected for further examination and comparison with the bacterial MTAN from Escherichia coli (EcMTAN). The structures, dynamics and thermodynamic properties of these enzymes were analyzed by X-ray crystallography, hydrogen-exchange coupled mass spectrometry and isothermal titration calorimetry, respectively. Our studies reveal that structural differences alone do not sufficiently explain the divergence in substrate specificity, and that conformational flexibility also plays an important role in substrate selection in MTANs. MTANs from the pathogenic bacterial species, Staphylococcus aureus and Streptococcus pneumoniae, were examined kinetically and structurally. Comparison of the structures and catalytic activities of these enzymes with EcMTAN shows that the discrepancies in kinetic activities arefully explained by structural differences, as the overall structure and active sites of these bacterial MTANs are nearly identical. These experiments are in agreement with our proposal that dynamics play a significant role in catalytic activity of MTAN, and suggest that both structure and dynamics must be considered in future antibiotic design. To further our understanding on the catalytic mechanism of MTAN, the putative catalytic residues of AtMTAN1 were identified by structural comparison to EcMTAN and mutated by site-directed mutagenesis. The AtMTAN1 mutants were analyzed by circular dichroism and kinetic studies. Our results suggest that the catalytic mechanism is largely conserved between bacterial and plant MTANs, although the role of the putative catalytic acid remains to be confirmed.

No description available.

Adenylates are metabolites with essential function in metabolism and signaling in all living organisms. As Cofactors, they enable thermodynamically unfavorable reactions to be catalyzed enzymatically within cells. Outside the cell, adenylates are involved in signalling processes in animals and emerging evidence suggests similar signaling mechanisms in the plants’ apoplast. Presumably, apoplastic apyrases are involved in this signaling by hydrolyzing the signal mediating molecules ATP and ADP to AMP. This PhD thesis focused on the role of adenylates on metabolism and development of potato (Solanum tuberosum) by using reverse genetics and biochemical approaches. To study the short and long term effect of cellular ATP and the adenylate energy charge on potato tuber metabolism, an apyrase from Escherichia coli targeted into the amyloplast was expressed inducibly and constitutively. Both approaches led to the identification of adaptations to reduced ATP/energy charge levels on the molecular and developmental level. These comprised a reduction of metabolites and pathway fluxes that require significant amounts of ATP, like amino acid or starch synthesis, and an activation of processes that produce ATP, like respiration and an immense increase in the surface-to-volume ratio. To identify extracellular enzymes involved in adenylate conversion, green fluorescent protein and activity localization studies in potato tissue were carried out. It was found that extracellular ATP is imported into the cell by an apoplastic enzyme complement consisting of apyrase, unspecific phosphatase, adenosine nucleosidase and an adenine transport system. By changing the expression of a potato specific apyrase via transgenic approaches, it was found that this enzyme has strong impact on plant and particular tuber development in potato. Whereas metabolite levels were hardly altered, transcript profiling of tubers with reduced apyrase activity revealed a significant upregulation of genes coding for extensins, which are associated with polar growth. The results are discussed in context of adaptive responses of plants to changes in the adenylate levels and the proposed role of apyrase in apoplastic purinergic signaling and ATP salvaging. In summary, this thesis provides insight into adenylate regulated processes within and outside non-photosynthetic plant cells. / Adenylate haben essentielle Funktionen in Stoffwechselprozessen und fungieren als Signalmoleküle in allen Organismen. Als Cofaktoren ermöglichen sie die Katalyse thermodynamisch ungünstiger Reaktionen innerhalb der Zelle, und außerhalb der Zelle wirken sie als Signalmoleküle in Tieren und nach neueren Forschungsergebnissen wohl auch in Pflanzen. Vermutlich wird die Signalwirkung von ATP und ADP durch Hydrolyse zu AMP unter Beteiligung apoplastische Apyrasen terminiert. Diese Arbeit behandelt den Einfluss der Adenylate auf Stoffwechsel- und Entwicklungsprozesse in der Kartoffelpflanze (Solanum tuberosum) mittels biochemischer und revers-genetischer Ansätze. Um kurzfristige und langfristige Einflüsse zellulären ATPs und der Energieladung auf den Stoffwechsel von Kartoffelknollen zu untersuchen, wurde eine mit einem plastidären Transitpeptid fusionierte Apyrase aus Escherichia coli induzierbar und dauerhaft exprimiert. Beide Ansätze führten zur Identifizierung von Anpassungen an eine reduzierte ATP Verfügbarkeit bzw. verringerte Energieladung. Die Anpassungen beinhalteten eine Reduzierung von ATP-verbrauchenden Stoffwechselaktivitäten und Stoffwechselprodukten, wie die Aminosäure- oder Stärkesynthese, und eine Aktivierung von Prozessen, welche die ATP-Bildung oder eine effizientere ATP-Bildung ermöglichen, wie Zellatmung und die Vergrößerung des Oberfächen/Volumen-Verhältnisses der Kartoffelknolle. Extrazelluläre Adenylat-umsetzende Enzyme wurden mit Hilfe des grün fluoreszierenden Proteins und Aktivitätsmessungen identifiziert und charakterisiert. Es wurde ein potentieller ATP Bergungsstoffwechselweg gefunden, der ATP über die Enzyme Apyrase, unspezifische Phosphatase und Adenosin-Nukleosidase zu Adenin umsetzt, welches über eine Purin-Permease in die Zelle transportiert wird. Transgene Manipulation der Aktivität der kartoffelspezifischen Apyrase zeigte, dass dieses Enzym einen großen Einfluss auf die Pflanzen-, insbesondere die Knollenentwicklung hat. Obwohl sich Stoffwechselaktivitäten kaum verändert hatten, führte die Verringerung der Apyrase Aktivität in den Knollen zur übermäßigen Expression von Extensin-Genen, die eine Funktion im polaren Wachstum von Pflanzenzellen besitzen. Die Ergebnisse wurden mit Hinblick auf Anpassungen der Pflanze an veränderte Adenylat-Spiegel und der potentiellen Beteiligung der endogenen Apyrase an einem apoplastischen ATP-Signalweg bzw. ATP-Bergungsstoffwechselweg diskutiert. Zusammengefasst, präsentiert diese Arbeit neue Einsichten in Adenylat-regulierte Prozesse in- und außerhalb nicht-photosynthetischer Pflanzenzellen.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.