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Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/26235

Title: Characterization of the Substrate Specificity and Catalytic Mechanism of 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase
Authors: Siu, Karen Ka Wing
Advisor: Howell, P. Lynne
Department: Biochemistry
Keywords: nucleosidase, substrate specificity, S-adenosylhomocysteine, 5'-Methylthioadenosine
Issue Date: 17-Feb-2011
Abstract: 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.
URI: http://hdl.handle.net/1807/26235
Appears in Collections:Doctoral

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