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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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry. Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis. In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries. By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis. MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis. / <p>QC 20161125</p>

Filamentous aeroterestrial green algae genus Klebsormidium occurs in a very wide range of freshwater and terrestrial habitats. Recent results of molecular investigations led to the finding that the diversity within this genus is far greater than expected on the basis of the morphological features, and that the traditional phenotypic species concept is insufficient. I tried to differentiate phylogenetic lineages within the genus Klebsormidium by thein different biogeographical distribution and environmental preferences. Since no study dealing with the biogeographic pattern of aeroterrestrial algae was so far undertaken, another aim of this work was to test validity of the protist ubiquity model in aeroterrestrial habitats. I studied this issues based on the chloroplast rbcL molecular marker. Based on the obtained data I found that the geographic definition of particular Klebsormidium lineages turns out to be unusable because of the cosmopolitan occurrence of almost all genotypes. However, the data obtained from the substrate specificity study shows that clear ecological preferences exist within the genus Klebsormidium and could be simply used to define different lineages within the genus.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 113-118).

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 139-159).