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In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from <i> Azotobactor vinelandii </i> complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. <p>In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources.

In this research project, structural aspects of monomeric NADP+-dependent isocitrate dehydrogenase from Corynebacterium glutamicum (CgIDH) are investigated together with site-directed mutagenesis and fluorescence spectroscopy studies. CgIDH, one of the enzymes of the Krebs cycle, catalyzes the decarboxylation of isocitrate into α-ketoglutarate, which in some bacteria and plants regulates the flow of carbon into either the Krebs cycle or the glyoxylate bypass depending on the available carbon source. The structure of CgIDH complexed with Mg2+ has been determined at 1.75 Å resolution using X-ray crystallography. In contrast to the closed conformation of published structures of monomeric NADP+-dependent IDH from <i> Azotobactor vinelandii </i> complexed with either isocitrate-Mn2+ or NADP+, the structure of CgIDH complexed with Mg2+ demonstrates the open conformation. The superimposed structure of CgIDH complexed with Mg2+ onto the structures of AvIDH complexes reveals that Domain II is rotated ~24° or ~35º, respectively, relative to Domain I when isocitrate-Mn2+ or NADP+ is bound, resulting in the closure of the active site between the two domains. Fluorescence spectroscopic studies support the proposal that the presence of isocitrate or NADP+ could mediate the conformational changes in CgIDH. <p>In addition, three CgIDH mutants (S130D, K253Q, and Y416T) were created based on the structural analysis and previous mutagenesis studies of homodimeric NADP+-dependent IDH. Both the specific activities and the fluorescence spectra of these CgIDH mutants elucidate the roles of these active site residues in CgIDH catalysis. It has been suggested that the conformational changes observed in the presence of the substrate(s) may regulate enzymatic activity in CgIDH, in contrast to homodimeric NADP+-dependent IDH in Escherichia coli, where the phosphorylation cycle controls activity. It is also presumed that both Lys253 and Tyr416 may play critical roles in CgIDH activity, as do the equivalent residues in homodimeric IDH from porcine heart mitochondria. Similar structural features and conformational changes among monomeric CgIDH and homodimeric NADP+-dependent IDH enzymes suggest the phylogenetic relationships among various monomeric and homodimeric NADP+-dependent IDH from different sources.

Metalloproteins represent a large portion of the total proteome. When bound to a protein a metal ion influences both protein stability and function through structural, catalytic or regulatory roles. Discovery of a metal ion cofactor presents new insight into both the structural and functional aspects of a protein and can be essential for the elucidation of the functional and mechanistic details of a protein of interest. The cupin, 2-oxoglutarate/Fe2+-dependent oxygenases (2OG oxygenases) and the di-iron oxygenase families of metalloproteins exemplify the diversity and catalytic potential of a metal ion cofactor, as well as the conservation of 3-dimensional fold and structural features in proteins with similar functions. The structural and biochemical characterization of three novel microbial metalloenzymes are presented; two Escherichia coli hypothetical proteins of previously unknown function, E. coli cupin sugar isomerase (EcSI) and a 2OG oxygenase, YcfD, and the novel microbial carbon-phosphorus (C-P) bond cleavage enzyme, PhnZ, are presented. In each case the identification of a metal ion cofactor and structure determination led to important functional insights. EcSI is encoded by a gene that is highly conserved among pathogenic bacteria. It has been identified as a sugar isomerase with specificity for the rare sugar D-lyxose as well as D-mannose based on structural homology to the cupin phosphoglucose isomerases, suggesting a role for EcSI in metabolism of alternative carbon sources. Structural homology of YcfD, the second metalloenzyme, to the 2OG oxygenase family, particularily human proteins involved in ribosome assembly, combined with evidence that YcfD interacts with the essential ribosomal protein L-16 provides the first evidence of translational regulation by a 2OG oxygenase in E. coli. The third metalloenzyme, PhnZ, was previously identified as an iron dependent oxygenase. Structural characterization revealed that PhnZ possesses a di-iron cofactor and shows significant structural homology to a di-iron oxygenase, providing structural evidence for its novel mechanism of C-P bond cleavage. Combined, these three structures also highlight several features of metal ion-enzyme interaction and regulation mechanisms employed by metalloenzymes as well as the importance of structure in the elucidation of functional and mechanistic characteristics of a protein. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-01-24 21:21:36.195

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A doença do sono é causada pelo parasita Tripanosoma brucei. Considerada uma doença negligenciada, mata milhares de pessoas todos os anos na África subsaariana. O T.brucei não apresenta resposta imune pronunciada, o que dificulta o desenvolvimento de vacinas, e os medicamentos disponíveis são escassos. Os tripanossomatídeos são comprovadamente sensíveis ao stress oxidativo causado pelo radical superóxido. Assim, as enzimas superóxido dismutases (SODs) são a primeira linha de defesa contra esse radical. As SODs são metalo enzimas (EC 1.5.1.1) capazes de catalisar a dismutação do superóxido em oxigênio molecular e peróxido de hidrogênio. São classificadas de acordo com o metal incorporado na estrutura: cobre e zinco (CuZnSOD), ferro ou manganês (Fe/MnSOD) e níquel (NiSODs). Neste trabalho de mestrado, a enzima TbFeSODB2 de T.brucei foi, expressa, purificada, cristalizada e teve sua estrutura resolvida. A estrutura cristalográfica da enzima do parasita foi comparada com a enzima humana análoga contendo manganês (HuMnSOD), onde foram evidenciadas as principais diferenças entre as duas estruturas que podem ser exploradas para o desenho do novos inibidores seletivos. Foi realizada uma análise de acoplamento estatístico, onde com base em um alinhamento múltiplo dessas enzimas determinou-se resíduos que são capazes de interferir na seletividade do metal incorporado e estado oligomérico das SODs. / Sleeping sickness, caused by the parasite Tripanosoma brucei, is considered a neglected disease, killing thousands of people every year in subsaharian Africa. T. brucei does not generate a pronounced immune response, difficulting the development of vaccines. Furthermore, available medicines are scarce. Tripanosomatides are known to be sensitive to oxidative stress caused by the superoxide radical. Therefore, the superoxide dismutase enzymes (SODs) are a primary line of defence for the parasites against this radical. SODs are metalloenzymes (EC 1.5.1.1) capable of catalyzing superoxide dismutation into molecular oxygen and hydrogen peroxide. SODs are classified according to the incorporated metal: copper/zinc (CuZnSOD), iron/manganese (Fe or MnSOD) and nickel (NiSODs). In the work presented here, TbFeSODB2 from T. brucei was expressed, purified, crystallized and its 3D structure solved. The crystal structure of the parasite enzyme was compared to the homologous human enzyme containing manganese (HuMnSOD), revealing evidence for differences between both structures which could be exploited in the design of new selective inhibitors. In addition, a statistical coupling analysis was performed on the entire Fe/MnSOD superfamily, based on a multiple sequence alignment. It was shown that this technique was able to identify novel residue determinants of metal selectivity and oligomeric state.

A doença do sono é causada pelo parasita Tripanosoma brucei. Considerada uma doença negligenciada, mata milhares de pessoas todos os anos na África subsaariana. O T.brucei não apresenta resposta imune pronunciada, o que dificulta o desenvolvimento de vacinas, e os medicamentos disponíveis são escassos. Os tripanossomatídeos são comprovadamente sensíveis ao stress oxidativo causado pelo radical superóxido. Assim, as enzimas superóxido dismutases (SODs) são a primeira linha de defesa contra esse radical. As SODs são metalo enzimas (EC 1.5.1.1) capazes de catalisar a dismutação do superóxido em oxigênio molecular e peróxido de hidrogênio. São classificadas de acordo com o metal incorporado na estrutura: cobre e zinco (CuZnSOD), ferro ou manganês (Fe/MnSOD) e níquel (NiSODs). Neste trabalho de mestrado, a enzima TbFeSODB2 de T.brucei foi, expressa, purificada, cristalizada e teve sua estrutura resolvida. A estrutura cristalográfica da enzima do parasita foi comparada com a enzima humana análoga contendo manganês (HuMnSOD), onde foram evidenciadas as principais diferenças entre as duas estruturas que podem ser exploradas para o desenho do novos inibidores seletivos. Foi realizada uma análise de acoplamento estatístico, onde com base em um alinhamento múltiplo dessas enzimas determinou-se resíduos que são capazes de interferir na seletividade do metal incorporado e estado oligomérico das SODs. / Sleeping sickness, caused by the parasite Tripanosoma brucei, is considered a neglected disease, killing thousands of people every year in subsaharian Africa. T. brucei does not generate a pronounced immune response, difficulting the development of vaccines. Furthermore, available medicines are scarce. Tripanosomatides are known to be sensitive to oxidative stress caused by the superoxide radical. Therefore, the superoxide dismutase enzymes (SODs) are a primary line of defence for the parasites against this radical. SODs are metalloenzymes (EC 1.5.1.1) capable of catalyzing superoxide dismutation into molecular oxygen and hydrogen peroxide. SODs are classified according to the incorporated metal: copper/zinc (CuZnSOD), iron/manganese (Fe or MnSOD) and nickel (NiSODs). In the work presented here, TbFeSODB2 from T. brucei was expressed, purified, crystallized and its 3D structure solved. The crystal structure of the parasite enzyme was compared to the homologous human enzyme containing manganese (HuMnSOD), revealing evidence for differences between both structures which could be exploited in the design of new selective inhibitors. In addition, a statistical coupling analysis was performed on the entire Fe/MnSOD superfamily, based on a multiple sequence alignment. It was shown that this technique was able to identify novel residue determinants of metal selectivity and oligomeric state.

In 2007, a gene homolog of CDO encoded by the gene Gm237 in the DUF164 family was identified as cysteamine dioxygenase (ADO). ADO is one of the only known thiol dioxygenases found in mammals. Both ADO and its partner cysteine dioxygenase (CDO) are non-heme iron dependent enzymes that play a crucial role in the biosynthesis of taruine/hypotaurine by insertion of a dioxygen molecule. However, ADO has been overshadowed by CDO as heavy research focus on CDO over the past decade has led to the elucidation of its structure and possible mechanistic properties. In an effort to further understand ADO’s mechanism and regulating role in vivo, this work will be focused on the mammalian hADO and trying to gain further insight on hADO’s structural features via crystallography work. Investigation of the crystallization parameters for hADO has elucidated several potential conditions. Detailed work on these crystallization parameters will be presented.