{"id":{"repo_id":"alabama","oai_identifier":"oai:ir.ua.edu:123456789/13140"},"canonical_url":"https://search.dev.ndltd.org/etd/alabama/oai:ir.ua.edu:123456789/13140","repository":{"repo_id":"alabama","name":"University of Alabama","base_url":"https://ir-api.ua.edu/oai/request"},"display":{"title":"Study of the Dynamics of Retaining GT-B Fold Glycosyltransferases Using Hydrogen Deuterium Exchange Mass Spectrometry","abstract":"Glycosyltransferases (GTs) facilitate the transfer of sugar moieties from activated sugardonors to various sugar acceptors. Structurally, GTs are classified as GT-A and GT-B folds.While the mechanism of inverting GT-Bs is well-established, the mechanism for retaining GT-Bsis debated. Proposed to involve a substrate-assisted mechanism, retaining GT-Bs require precisedonor and acceptor substrate positioning. To achieve this, GT-Bs show global dynamics involvingrigid body rotation between two globular Rossmann domains and likely local dynamics. Toelucidate substrate-induced local dynamics, hydrogen-deuterium exchange mass spectrometry(HDX-MS) is used to analyze CgMshA from Corynebacterium glutamicum, a model enzymefrom GT-B fold. In the presence of acceptor substrate inositol-1-phosphate (I1P), decreaseddynamics occur in CgMshA's N-terminal domain (NTD) and linker regions. Conversely, withdonor substrate uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), dynamics decrease inboth domain halves. Identical deuterium uptake plots at the NTD with I1P or UDP-GlcNAcsuggest that UDP-GlcNAc binding creates the binding site for I1P. Surprisingly, UDP bindingresults in diminished dynamics in most NTD and C-terminal domain (CTD) residues.UDP-GlcNAc binding induces EX1 exchange kinetics at the CTD, absent with UDP. This studyunderscores the essential role of donor sugar moieties in enzyme dynamics. To delve into GT-Breaction mechanisms, evolutionarily conserved residues in the retaining GT-B fold enzymes fromthe GT4 family are investigated, which identified a conserved histidine residue on the NTD. Thishistidine residue is found to regulate substrate binding and ensure structural integrity. Observedonly in retaining GT-B enzymes, this histidine residue distinguishes retaining GT-B frominverting GT-B fold enzymes. This conserved histidine residue may be important for bindingdonor substrate in the bent-back conformation —a prerequisite for the productivesubstrate-assisted mechanism. The effect of molecular crowding on the catalysis of the retainingGT-B is explored, revealing varied effects. Not all crowding molecules uniformly influencecatalytic rates, with kinetic parameter analysis suggesting a minor role of volume exclusion.Conversely, crowder-substrate or crowder-protein interactions play a significant role in alteringkinetic parameters. In summary, these studies contribute to comprehending the mechanisms ofretaining GT-B glycosyltransferases.","abstract_html":"Glycosyltransferases (GTs) facilitate the transfer of sugar moieties from activated sugardonors to various sugar acceptors. Structurally, GTs are classified as GT-A and GT-B folds.While the mechanism of inverting GT-Bs is well-established, the mechanism for retaining GT-Bsis debated. Proposed to involve a substrate-assisted mechanism, retaining GT-Bs require precisedonor and acceptor substrate positioning. To achieve this, GT-Bs show global dynamics involvingrigid body rotation between two globular Rossmann domains and likely local dynamics. Toelucidate substrate-induced local dynamics, hydrogen-deuterium exchange mass spectrometry(HDX-MS) is used to analyze CgMshA from Corynebacterium glutamicum, a model enzymefrom GT-B fold. In the presence of acceptor substrate inositol-1-phosphate (I1P), decreaseddynamics occur in CgMshA's N-terminal domain (NTD) and linker regions. Conversely, withdonor substrate uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), dynamics decrease inboth domain halves. Identical deuterium uptake plots at the NTD with I1P or UDP-GlcNAcsuggest that UDP-GlcNAc binding creates the binding site for I1P. Surprisingly, UDP bindingresults in diminished dynamics in most NTD and C-terminal domain (CTD) residues.UDP-GlcNAc binding induces EX1 exchange kinetics at the CTD, absent with UDP. This studyunderscores the essential role of donor sugar moieties in enzyme dynamics. To delve into GT-Breaction mechanisms, evolutionarily conserved residues in the retaining GT-B fold enzymes fromthe GT4 family are investigated, which identified a conserved histidine residue on the NTD. Thishistidine residue is found to regulate substrate binding and ensure structural integrity. Observedonly in retaining GT-B enzymes, this histidine residue distinguishes retaining GT-B frominverting GT-B fold enzymes. This conserved histidine residue may be important for bindingdonor substrate in the bent-back conformation —a prerequisite for the productivesubstrate-assisted mechanism. The effect of molecular crowding on the catalysis of the retainingGT-B is explored, revealing varied effects. Not all crowding molecules uniformly influencecatalytic rates, with kinetic parameter analysis suggesting a minor role of volume exclusion.Conversely, crowder-substrate or crowder-protein interactions play a significant role in alteringkinetic parameters. In summary, these studies contribute to comprehending the mechanisms ofretaining GT-B glycosyltransferases.","abstract_has_math":false,"creators":["Karki, Ramesh"],"institution":"University of Alabama Libraries","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Dunkle, Jack","Woski, Stephen","Street, Shane","Ciesla, Lukasz"],"advisors":["Frantom, Patrick"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023","date_published":"2023","updated_at":"2026-07-27T18:44:23Z","subjects":[],"languages":["en_US","English"],"rights":["All rights reserved by the author unless otherwise indicated."],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["1026689"],"render_values":[{"text":"1026689","href":null,"code":true}]}]},"links":{"outbound_url":"https://ir.ua.edu/handle/123456789/13140","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Frantom, Patrick","Dunkle, Jack","Woski, Stephen","Street, Shane","Ciesla, Lukasz"]},{"key":"dc:contributor.advisor","label":"Advisor","values":["Frantom, Patrick"]},{"key":"dc:creator","label":"Author","values":["Karki, Ramesh"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2024-02-20T16:34:18Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-02-20T16:34:18Z"]},{"key":"dc:date.issued","label":"Date","values":["2023"]},{"key":"dc:publisher","label":"Institution","values":["University of Alabama Libraries"]},{"key":"dc:type","label":"Dc Type","values":["thesis","text"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English"]},{"key":"dc:language.iso","label":"Language (ISO)","values":["en_US"]},{"key":"dc:rights","label":"Dc Rights","values":["All rights reserved by the author unless otherwise indicated."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["1026689"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://ir.ua.edu/handle/123456789/13140"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Electronic Thesis or Dissertation"]},{"key":"dc:description.abstract","label":"Abstract","values":["Glycosyltransferases (GTs) facilitate the transfer of sugar moieties from activated sugardonors to various sugar acceptors. Structurally, GTs are classified as GT-A and GT-B folds.While the mechanism of inverting GT-Bs is well-established, the mechanism for retaining GT-Bsis debated. Proposed to involve a substrate-assisted mechanism, retaining GT-Bs require precisedonor and acceptor substrate positioning. To achieve this, GT-Bs show global dynamics involvingrigid body rotation between two globular Rossmann domains and likely local dynamics. Toelucidate substrate-induced local dynamics, hydrogen-deuterium exchange mass spectrometry(HDX-MS) is used to analyze CgMshA from Corynebacterium glutamicum, a model enzymefrom GT-B fold. In the presence of acceptor substrate inositol-1-phosphate (I1P), decreaseddynamics occur in CgMshA's N-terminal domain (NTD) and linker regions. Conversely, withdonor substrate uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), dynamics decrease inboth domain halves. Identical deuterium uptake plots at the NTD with I1P or UDP-GlcNAcsuggest that UDP-GlcNAc binding creates the binding site for I1P. Surprisingly, UDP bindingresults in diminished dynamics in most NTD and C-terminal domain (CTD) residues.UDP-GlcNAc binding induces EX1 exchange kinetics at the CTD, absent with UDP. This studyunderscores the essential role of donor sugar moieties in enzyme dynamics. To delve into GT-Breaction mechanisms, evolutionarily conserved residues in the retaining GT-B fold enzymes fromthe GT4 family are investigated, which identified a conserved histidine residue on the NTD. Thishistidine residue is found to regulate substrate binding and ensure structural integrity. Observedonly in retaining GT-B enzymes, this histidine residue distinguishes retaining GT-B frominverting GT-B fold enzymes. This conserved histidine residue may be important for bindingdonor substrate in the bent-back conformation —a prerequisite for the productivesubstrate-assisted mechanism. The effect of molecular crowding on the catalysis of the retainingGT-B is explored, revealing varied effects. Not all crowding molecules uniformly influencecatalytic rates, with kinetic parameter analysis suggesting a minor role of volume exclusion.Conversely, crowder-substrate or crowder-protein interactions play a significant role in alteringkinetic parameters. In summary, these studies contribute to comprehending the mechanisms ofretaining GT-B glycosyltransferases."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["electronic"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Study of the Dynamics of Retaining GT-B Fold Glycosyltransferases Using Hydrogen Deuterium Exchange Mass Spectrometry"]}]}],"canonical_facts":{"dc:contributor":["Frantom, Patrick","Dunkle, Jack","Woski, Stephen","Street, Shane","Ciesla, Lukasz"],"dc:contributor.advisor":["Frantom, Patrick"],"dc:creator":["Karki, Ramesh"],"dc:date.accessioned":["2024-02-20T16:34:18Z"],"dc:date.available":["2024-02-20T16:34:18Z"],"dc:date.issued":["2023"],"dc:description":["Electronic Thesis or Dissertation"],"dc:description.abstract":["Glycosyltransferases (GTs) facilitate the transfer of sugar moieties from activated sugardonors to various sugar acceptors. Structurally, GTs are classified as GT-A and GT-B folds.While the mechanism of inverting GT-Bs is well-established, the mechanism for retaining GT-Bsis debated. Proposed to involve a substrate-assisted mechanism, retaining GT-Bs require precisedonor and acceptor substrate positioning. To achieve this, GT-Bs show global dynamics involvingrigid body rotation between two globular Rossmann domains and likely local dynamics. Toelucidate substrate-induced local dynamics, hydrogen-deuterium exchange mass spectrometry(HDX-MS) is used to analyze CgMshA from Corynebacterium glutamicum, a model enzymefrom GT-B fold. In the presence of acceptor substrate inositol-1-phosphate (I1P), decreaseddynamics occur in CgMshA's N-terminal domain (NTD) and linker regions. Conversely, withdonor substrate uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), dynamics decrease inboth domain halves. Identical deuterium uptake plots at the NTD with I1P or UDP-GlcNAcsuggest that UDP-GlcNAc binding creates the binding site for I1P. Surprisingly, UDP bindingresults in diminished dynamics in most NTD and C-terminal domain (CTD) residues.UDP-GlcNAc binding induces EX1 exchange kinetics at the CTD, absent with UDP. This studyunderscores the essential role of donor sugar moieties in enzyme dynamics. To delve into GT-Breaction mechanisms, evolutionarily conserved residues in the retaining GT-B fold enzymes fromthe GT4 family are investigated, which identified a conserved histidine residue on the NTD. Thishistidine residue is found to regulate substrate binding and ensure structural integrity. Observedonly in retaining GT-B enzymes, this histidine residue distinguishes retaining GT-B frominverting GT-B fold enzymes. This conserved histidine residue may be important for bindingdonor substrate in the bent-back conformation —a prerequisite for the productivesubstrate-assisted mechanism. The effect of molecular crowding on the catalysis of the retainingGT-B is explored, revealing varied effects. Not all crowding molecules uniformly influencecatalytic rates, with kinetic parameter analysis suggesting a minor role of volume exclusion.Conversely, crowder-substrate or crowder-protein interactions play a significant role in alteringkinetic parameters. In summary, these studies contribute to comprehending the mechanisms ofretaining GT-B glycosyltransferases."],"dc:format.medium":["electronic"],"dc:format.mimetype":["application/pdf"],"dc:identifier.other":["1026689"],"dc:identifier.uri":["https://ir.ua.edu/handle/123456789/13140"],"dc:language":["English"],"dc:language.iso":["en_US"],"dc:publisher":["University of Alabama Libraries"],"dc:rights":["All rights reserved by the author unless otherwise indicated."],"dc:title":["Study of the Dynamics of Retaining GT-B Fold Glycosyltransferases Using Hydrogen Deuterium Exchange Mass Spectrometry"],"dc:type":["thesis","text"]},"updated_at":"2026-07-27T18:44:23Z"}