{"id":{"repo_id":"uwo","oai_identifier":"oai:uwo.scholaris.ca:20.500.14721/31737"},"canonical_url":"https://search.dev.ndltd.org/etd/uwo/oai:uwo.scholaris.ca:20.500.14721/31737","repository":{"repo_id":"uwo","name":"Western University","base_url":"https://uwo.scholaris.ca/server/oai/request"},"display":{"title":"Characterization of Enzymes in the L-Asparagine Metabolism Pathway in Legumes","abstract":"Improving the quality of economically and nutritionally valuable legume crops, such as soybean, requires a comprehensive understanding of nitrogen metabolism. L-asparagine (Asn) is an important source of nitrogen stored and transported in higher plants, favoured in legumes due to its advantageous 2N:4C ratio. The catabolism of Asn in tissues occurs through two major pathways: deamidation and transamination. The transamination pathway employs an unidentified α-ketosuccinamate (α-KSM ) reducing dehydrogenase (α-KSDH), as well as an ω-amidase which has not been fully characterized. Additionally, α-ketosuccinamate and α-hydroxysuccinamate are potentially toxic metabolites generated in this pathway. The goal of this research is to identify α-KSDH in soybean and characterize the role of ω-amidase in the transamination pathway in Arabidopsis. Fractionation, ammonium sulfate precipitation, size exclusion chromatography, and LC-MS/MS were employed to sequentially purify and identify an α-KSDH that showed activity with α-KSM. Altogether, these techniques revealed that soybean hydroxyphenylpyruvate reductase (GmHPPR) and Arabidopsis hydroxypyruvate reductase 2 (AtHPR2) catalyze the reduction of α-KSM into α-HSM. Based on this activity, these are likely the enzymes responsible for α-KSDH function in vivo of their respective species, however in vivo studies are required to confirm this function. The broad substrate specificity of HPPR and HPR2 and its activity with 2-hydroxyacids indicate that its main role in vivo is to convert intermediary metabolites into metabolites that can be directly used for other important pathways within the plant. An ω-amidase T-DNA insertion loss of function mutant was found to be embryo lethal in Arabidopsis, cause shortened siliques in heterozygotes, and overall have a detrimental effect on reproduction. The mechanism of these effects is likely a shortage of methionine and polyamines, toxic metabolite accumulation, or a combination of the two. Like ω-amidase, HPPR is known as a “clean-up” enzyme, and it can be assumed that it also plays a role in mitigating the levels of toxic intermediary metabolites as a “repair” enzyme. This research has advanced our understanding of Asn metabolism in higher plants and may ultimately contribute to advancements in crop nitrogen use efficiency.","abstract_html":"Improving the quality of economically and nutritionally valuable legume crops, such as soybean, requires a comprehensive understanding of nitrogen metabolism. L-asparagine (Asn) is an important source of nitrogen stored and transported in higher plants, favoured in legumes due to its advantageous 2N:4C ratio. The catabolism of Asn in tissues occurs through two major pathways: deamidation and transamination. The transamination pathway employs an unidentified α-ketosuccinamate (α-KSM ) reducing dehydrogenase (α-KSDH), as well as an ω-amidase which has not been fully characterized. Additionally, α-ketosuccinamate and α-hydroxysuccinamate are potentially toxic metabolites generated in this pathway. The goal of this research is to identify α-KSDH in soybean and characterize the role of ω-amidase in the transamination pathway in Arabidopsis. Fractionation, ammonium sulfate precipitation, size exclusion chromatography, and LC-MS/MS were employed to sequentially purify and identify an α-KSDH that showed activity with α-KSM. Altogether, these techniques revealed that soybean hydroxyphenylpyruvate reductase (GmHPPR) and Arabidopsis hydroxypyruvate reductase 2 (AtHPR2) catalyze the reduction of α-KSM into α-HSM. Based on this activity, these are likely the enzymes responsible for α-KSDH function in vivo of their respective species, however in vivo studies are required to confirm this function. The broad substrate specificity of HPPR and HPR2 and its activity with 2-hydroxyacids indicate that its main role in vivo is to convert intermediary metabolites into metabolites that can be directly used for other important pathways within the plant. An ω-amidase T-DNA insertion loss of function mutant was found to be embryo lethal in Arabidopsis, cause shortened siliques in heterozygotes, and overall have a detrimental effect on reproduction. The mechanism of these effects is likely a shortage of methionine and polyamines, toxic metabolite accumulation, or a combination of the two. Like ω-amidase, HPPR is known as a “clean-up” enzyme, and it can be assumed that it also plays a role in mitigating the levels of toxic intermediary metabolites as a “repair” enzyme. This research has advanced our understanding of Asn metabolism in higher plants and may ultimately contribute to advancements in crop nitrogen use efficiency.","abstract_has_math":false,"creators":["Zaman, Raiyan D"],"institution":"The University of Western Ontario","degree_name":"Ph D","degree_level":null,"degree_discipline":"Biology","degree_department":null,"school":null,"contributors":[],"advisors":["Marsolais, Frédéric","Bernards, Mark"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-03-13","date_published":"2025-03-13","updated_at":"2026-07-27T21:55:56Z","subjects":["asparagine metabolism","α-ketosuccinamate","α-hydroxysuccinamate","hydroxypyruvate reductase","hydroxyphenylpyruvate reductase","ω-amidase"],"languages":["en_ca"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/20.500.14721/31737","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Marsolais, Frédéric","Bernards, Mark"]},{"key":"dc:creator","label":"Author","values":["Zaman, Raiyan D"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-07-10T19:21:35Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-03-13"]},{"key":"dc:publisher","label":"Institution","values":["The University of Western Ontario"]},{"key":"dc:type","label":"Dc Type","values":["thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biology"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph D"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["asparagine metabolism","α-ketosuccinamate","α-hydroxysuccinamate","hydroxypyruvate reductase","hydroxyphenylpyruvate reductase","ω-amidase"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_ca"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/20.500.14721/31737"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Collaborative Specialization: Scientific Computing","The thesis cover page in the PDF document includes references to Western University’s previous institutional repository platform, known as Scholarship@Western, and links to that platform (beginning with ir.lib.uwo.ca). In citing or referring to this thesis, use the DOI or handle from this page instead. Sample citation: Author name, \"Thesis title.\" (Year). Western University Open Repository. https://doi.org/10.71858/123456."]},{"key":"dc:description.abstract","label":"Abstract","values":["Improving the quality of economically and nutritionally valuable legume crops, such as soybean, requires a comprehensive understanding of nitrogen metabolism. L-asparagine (Asn) is an important source of nitrogen stored and transported in higher plants, favoured in legumes due to its advantageous 2N:4C ratio. The catabolism of Asn in tissues occurs through two major pathways: deamidation and transamination. The transamination pathway employs an unidentified α-ketosuccinamate (α-KSM ) reducing dehydrogenase (α-KSDH), as well as an ω-amidase which has not been fully characterized. Additionally, α-ketosuccinamate and α-hydroxysuccinamate are potentially toxic metabolites generated in this pathway. The goal of this research is to identify α-KSDH in soybean and characterize the role of ω-amidase in the transamination pathway in Arabidopsis. Fractionation, ammonium sulfate precipitation, size exclusion chromatography, and LC-MS/MS were employed to sequentially purify and identify an α-KSDH that showed activity with α-KSM. Altogether, these techniques revealed that soybean hydroxyphenylpyruvate reductase (GmHPPR) and Arabidopsis hydroxypyruvate reductase 2 (AtHPR2) catalyze the reduction of α-KSM into α-HSM. Based on this activity, these are likely the enzymes responsible for α-KSDH function in vivo of their respective species, however in vivo studies are required to confirm this function. The broad substrate specificity of HPPR and HPR2 and its activity with 2-hydroxyacids indicate that its main role in vivo is to convert intermediary metabolites into metabolites that can be directly used for other important pathways within the plant. An ω-amidase T-DNA insertion loss of function mutant was found to be embryo lethal in Arabidopsis, cause shortened siliques in heterozygotes, and overall have a detrimental effect on reproduction. The mechanism of these effects is likely a shortage of methionine and polyamines, toxic metabolite accumulation, or a combination of the two. Like ω-amidase, HPPR is known as a “clean-up” enzyme, and it can be assumed that it also plays a role in mitigating the levels of toxic intermediary metabolites as a “repair” enzyme. This research has advanced our understanding of Asn metabolism in higher plants and may ultimately contribute to advancements in crop nitrogen use efficiency."]},{"key":"dc:title","label":"Title","values":["Characterization of Enzymes in the L-Asparagine Metabolism Pathway in Legumes"]}]}],"canonical_facts":{"dc:contributor.advisor":["Marsolais, Frédéric","Bernards, Mark"],"dc:creator":["Zaman, Raiyan D"],"dc:date.accessioned":["2025-07-10T19:21:35Z"],"dc:date.issued":["2025-03-13"],"dc:description":["Collaborative Specialization: Scientific Computing","The thesis cover page in the PDF document includes references to Western University’s previous institutional repository platform, known as Scholarship@Western, and links to that platform (beginning with ir.lib.uwo.ca). In citing or referring to this thesis, use the DOI or handle from this page instead. Sample citation: Author name, \"Thesis title.\" (Year). Western University Open Repository. https://doi.org/10.71858/123456."],"dc:description.abstract":["Improving the quality of economically and nutritionally valuable legume crops, such as soybean, requires a comprehensive understanding of nitrogen metabolism. L-asparagine (Asn) is an important source of nitrogen stored and transported in higher plants, favoured in legumes due to its advantageous 2N:4C ratio. The catabolism of Asn in tissues occurs through two major pathways: deamidation and transamination. The transamination pathway employs an unidentified α-ketosuccinamate (α-KSM ) reducing dehydrogenase (α-KSDH), as well as an ω-amidase which has not been fully characterized. Additionally, α-ketosuccinamate and α-hydroxysuccinamate are potentially toxic metabolites generated in this pathway. The goal of this research is to identify α-KSDH in soybean and characterize the role of ω-amidase in the transamination pathway in Arabidopsis. Fractionation, ammonium sulfate precipitation, size exclusion chromatography, and LC-MS/MS were employed to sequentially purify and identify an α-KSDH that showed activity with α-KSM. Altogether, these techniques revealed that soybean hydroxyphenylpyruvate reductase (GmHPPR) and Arabidopsis hydroxypyruvate reductase 2 (AtHPR2) catalyze the reduction of α-KSM into α-HSM. Based on this activity, these are likely the enzymes responsible for α-KSDH function in vivo of their respective species, however in vivo studies are required to confirm this function. The broad substrate specificity of HPPR and HPR2 and its activity with 2-hydroxyacids indicate that its main role in vivo is to convert intermediary metabolites into metabolites that can be directly used for other important pathways within the plant. An ω-amidase T-DNA insertion loss of function mutant was found to be embryo lethal in Arabidopsis, cause shortened siliques in heterozygotes, and overall have a detrimental effect on reproduction. The mechanism of these effects is likely a shortage of methionine and polyamines, toxic metabolite accumulation, or a combination of the two. Like ω-amidase, HPPR is known as a “clean-up” enzyme, and it can be assumed that it also plays a role in mitigating the levels of toxic intermediary metabolites as a “repair” enzyme. This research has advanced our understanding of Asn metabolism in higher plants and may ultimately contribute to advancements in crop nitrogen use efficiency."],"dc:identifier.uri":["https://hdl.handle.net/20.500.14721/31737"],"dc:language.iso":["en_ca"],"dc:publisher":["The University of Western Ontario"],"dc:subject":["asparagine metabolism","α-ketosuccinamate","α-hydroxysuccinamate","hydroxypyruvate reductase","hydroxyphenylpyruvate reductase","ω-amidase"],"dc:title":["Characterization of Enzymes in the L-Asparagine Metabolism Pathway in Legumes"],"dc:type":["thesis"],"thesis:degree_discipline":["Biology"],"thesis:degree_name":["Ph D"]},"updated_at":"2026-07-27T21:55:56Z"}