Study of the Dynamics of Retaining GT-B Fold Glycosyltransferases Using Hydrogen Deuterium Exchange Mass Spectrometry

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.

Grantor dc:publisher

University of Alabama Libraries

Year dc:date.issued

2023

Author dc:creator

• Karki, Ramesh

Advisor dc:contributor.advisor

• Frantom, Patrick

Contributors dc:contributor

• Dunkle, Jack
• Woski, Stephen
• Street, Shane
• Ciesla, Lukasz

Statement dc:rights

• All rights reserved by the author unless otherwise indicated.

Language dc:language.iso

en_US, English

Dc Identifier Other

1026689

OAI identifier oai:identifier

oai:ir.ua.edu:123456789/13140