{"id":{"repo_id":"toronto-retro","oai_identifier":"oai:utoronto.scholaris.ca:1807/102758"},"canonical_url":"https://search.dev.ndltd.org/etd/toronto-retro/oai:utoronto.scholaris.ca:1807/102758","repository":{"repo_id":"toronto-retro","name":"University of Toronto","base_url":"https://utoronto.scholaris.ca/server/oai/request"},"display":{"title":"Investigation of Escherichia coli [NiFe]-Hydrogenase Maturation","abstract":"[NiFe]-hydrogenases catalyze the reversible oxidation of hydrogen gas at a bimetallic active site and are important enzymes in bacteria and archaea for anaerobic growth and pathogenesis. The maturation of [NiFe]-hydrogenase requires at least seven dedicated accessory proteins to assemble and insert the components of the NiFe(CN)2CO catalytic site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase precursor protein, a process that is accomplished by the metallochaperone proteins HypA, HypB, and SlyD. This delivery process is supported by proteins that import, export, regulate, and store nickel to ensure a sufficient supply while also mitigating the innate toxicity of this transition metal. In this work, nickel delivery to the [NiFe]-hydrogenase in Escherichia coli was examined using microbiological, biochemical, spectroscopic, and computational methods. The results demonstrate that protein-protein interactions between the metallochaperones afford layers of nickel selectivity. The protein complexes HypA-HypB and SlyD-HypB are modulated by the GTPase cycle of HypB and mediate the selective release of nickel over zinc. In addition, characterization of the metal-binding sites of HypA and HypB revealed different binding modalities, suggesting distinct acquisition and release mechanisms for these metallochaperones. This thesis also describes the development and employment of a high-throughput whole-cell [NiFe]-hydrogenase assay. The assay was used to screen the Keio collection of single gene deletion strains of E. coli and uncovered, for the first time, eutK as a component of the nickel delivery pathway and nickel homeostasis. The assay was also used to determine extracellular nickel concentrations required to bypass the nickel uptake and delivery processes to validate those systems as potential therapeutic targets, setting the stage for the search for small molecules that inhibit the biosynthesis of [NiFe]-hydrogenase. Together, this work gives insight into several aspects of the crucial nickel delivery process that bacteria use to produce [NiFe]-hydrogenase.","abstract_html":"[NiFe]-hydrogenases catalyze the reversible oxidation of hydrogen gas at a bimetallic active site and are important enzymes in bacteria and archaea for anaerobic growth and pathogenesis. The maturation of [NiFe]-hydrogenase requires at least seven dedicated accessory proteins to assemble and insert the components of the NiFe(CN)2CO catalytic site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase precursor protein, a process that is accomplished by the metallochaperone proteins HypA, HypB, and SlyD. This delivery process is supported by proteins that import, export, regulate, and store nickel to ensure a sufficient supply while also mitigating the innate toxicity of this transition metal. In this work, nickel delivery to the [NiFe]-hydrogenase in Escherichia coli was examined using microbiological, biochemical, spectroscopic, and computational methods. The results demonstrate that protein-protein interactions between the metallochaperones afford layers of nickel selectivity. The protein complexes HypA-HypB and SlyD-HypB are modulated by the GTPase cycle of HypB and mediate the selective release of nickel over zinc. In addition, characterization of the metal-binding sites of HypA and HypB revealed different binding modalities, suggesting distinct acquisition and release mechanisms for these metallochaperones. This thesis also describes the development and employment of a high-throughput whole-cell [NiFe]-hydrogenase assay. The assay was used to screen the Keio collection of single gene deletion strains of E. coli and uncovered, for the first time, eutK as a component of the nickel delivery pathway and nickel homeostasis. The assay was also used to determine extracellular nickel concentrations required to bypass the nickel uptake and delivery processes to validate those systems as potential therapeutic targets, setting the stage for the search for small molecules that inhibit the biosynthesis of [NiFe]-hydrogenase. Together, this work gives insight into several aspects of the crucial nickel delivery process that bacteria use to produce [NiFe]-hydrogenase.","abstract_has_math":false,"creators":["Lacasse, Michael Joseph"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Chemistry","school":null,"contributors":[],"advisors":["Zamble, Deborah B"],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-11","date_published":"2019-11","updated_at":"2026-07-27T21:27:54Z","subjects":["Escherichia coli","Hydrogenase","Metal Homeostasis","Metallochaperones","Nickel"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1807/102758","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Zamble, Deborah B"]},{"key":"dc:contributor.department","label":"Department","values":["Chemistry"]},{"key":"dc:creator","label":"Author","values":["Lacasse, Michael Joseph"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-11"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2020-11-13T05:00:39Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2020-11-13T05:00:39Z"]},{"key":"dc:date.issued","label":"Date","values":["2019-11"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Escherichia coli","Hydrogenase","Metal Homeostasis","Metallochaperones","Nickel"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1807/102758"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["[NiFe]-hydrogenases catalyze the reversible oxidation of hydrogen gas at a bimetallic active site and are important enzymes in bacteria and archaea for anaerobic growth and pathogenesis. The maturation of [NiFe]-hydrogenase requires at least seven dedicated accessory proteins to assemble and insert the components of the NiFe(CN)2CO catalytic site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase precursor protein, a process that is accomplished by the metallochaperone proteins HypA, HypB, and SlyD. This delivery process is supported by proteins that import, export, regulate, and store nickel to ensure a sufficient supply while also mitigating the innate toxicity of this transition metal. In this work, nickel delivery to the [NiFe]-hydrogenase in Escherichia coli was examined using microbiological, biochemical, spectroscopic, and computational methods. The results demonstrate that protein-protein interactions between the metallochaperones afford layers of nickel selectivity. The protein complexes HypA-HypB and SlyD-HypB are modulated by the GTPase cycle of HypB and mediate the selective release of nickel over zinc. In addition, characterization of the metal-binding sites of HypA and HypB revealed different binding modalities, suggesting distinct acquisition and release mechanisms for these metallochaperones. This thesis also describes the development and employment of a high-throughput whole-cell [NiFe]-hydrogenase assay. The assay was used to screen the Keio collection of single gene deletion strains of E. coli and uncovered, for the first time, eutK as a component of the nickel delivery pathway and nickel homeostasis. The assay was also used to determine extracellular nickel concentrations required to bypass the nickel uptake and delivery processes to validate those systems as potential therapeutic targets, setting the stage for the search for small molecules that inhibit the biosynthesis of [NiFe]-hydrogenase. Together, this work gives insight into several aspects of the crucial nickel delivery process that bacteria use to produce [NiFe]-hydrogenase."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Investigation of Escherichia coli [NiFe]-Hydrogenase Maturation"]}]}],"canonical_facts":{"dc:contributor.advisor":["Zamble, Deborah B"],"dc:contributor.department":["Chemistry"],"dc:creator":["Lacasse, Michael Joseph"],"dc:date":["2019-11"],"dc:date.accessioned":["2020-11-13T05:00:39Z"],"dc:date.available":["2020-11-13T05:00:39Z"],"dc:date.issued":["2019-11"],"dc:description.abstract":["[NiFe]-hydrogenases catalyze the reversible oxidation of hydrogen gas at a bimetallic active site and are important enzymes in bacteria and archaea for anaerobic growth and pathogenesis. The maturation of [NiFe]-hydrogenase requires at least seven dedicated accessory proteins to assemble and insert the components of the NiFe(CN)2CO catalytic site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase precursor protein, a process that is accomplished by the metallochaperone proteins HypA, HypB, and SlyD. This delivery process is supported by proteins that import, export, regulate, and store nickel to ensure a sufficient supply while also mitigating the innate toxicity of this transition metal. In this work, nickel delivery to the [NiFe]-hydrogenase in Escherichia coli was examined using microbiological, biochemical, spectroscopic, and computational methods. The results demonstrate that protein-protein interactions between the metallochaperones afford layers of nickel selectivity. The protein complexes HypA-HypB and SlyD-HypB are modulated by the GTPase cycle of HypB and mediate the selective release of nickel over zinc. In addition, characterization of the metal-binding sites of HypA and HypB revealed different binding modalities, suggesting distinct acquisition and release mechanisms for these metallochaperones. This thesis also describes the development and employment of a high-throughput whole-cell [NiFe]-hydrogenase assay. The assay was used to screen the Keio collection of single gene deletion strains of E. coli and uncovered, for the first time, eutK as a component of the nickel delivery pathway and nickel homeostasis. The assay was also used to determine extracellular nickel concentrations required to bypass the nickel uptake and delivery processes to validate those systems as potential therapeutic targets, setting the stage for the search for small molecules that inhibit the biosynthesis of [NiFe]-hydrogenase. Together, this work gives insight into several aspects of the crucial nickel delivery process that bacteria use to produce [NiFe]-hydrogenase."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1807/102758"],"dc:subject":["Escherichia coli","Hydrogenase","Metal Homeostasis","Metallochaperones","Nickel"],"dc:title":["Investigation of Escherichia coli [NiFe]-Hydrogenase Maturation"],"dc:type":["Thesis"]},"updated_at":"2026-07-27T21:27:54Z"}