Master Thesis: Computational study of GumH - A retaining glycosyltransferase involved in biosynthesis of xanthan gum

Abstract: Xanthan gum is an industrially important polysaccharide with diverse applications and has shown promise as a sustainable alternative to materials based on fossil-derived or otherwise environmentally harmful compounds. Despite its widespread use, there is still interest in further expanding its potential through structural modification. By modifying enzymes in a controlled way, enzyme engineering could make it possible to produce XG with alternative structures, possibly also using fewer resources in the production. However, it requires detailed knowledge of the proteins. Xanthan gum is synthesized by twelve GTs located in or near the inner membrane of X. campestris. This thesis aimed to investigate the structure, function, and dynamic behavior of GumH using computational methods, with the final objective of enabling enzymatic engineering of the xanthan gum side chain. GumH catalyzes the addition of the first mannose residue in the xanthan gum side chain. Its structure is not solved; however, based on its function and sequence, it has been categorized as a retaining GT from family 4 that adopts the GT-B fold. The mechanisms of retaining GTs are difficult to establish using classical experimental methods, and as a result, are not fully understood, and so neither the mechanism nor the structure of this protein is known. GumH is a membrane protein. Due to its structural stability observed in simulations with and without a membrane, along with OPM predictions and the chemical properties of motifs in the acceptor domain, we hypothesize that GumH, more specifically, is an integral membrane protein. GumH's dynamics were analyzed using PCA of MD trajectories and NMA. The key functional motion was identified as a domain rearrangement in which the donor domain rotates toward the membrane, resulting in a closed conformation and assembly of the enzyme’s active site. This is consistent with the dynamic behavior observed in other GTs. The equilibrium state of GumH appears to be the open conformation. Simulations indicated that a transient closed conformation can occur without substrates; however, binding of both donor and acceptor substrates promotes a stable closed fold. MD simulations enabled the identification of residues involved in substrate binding. Twelve residues were found to interact with the donor substrate, and eight with the lipidic acceptor substrate. Of these, five (Gly16, Arg198, Lys203, Glu279, Glu287) have been reported as essential for enzymatic activity in other GTs. Furthermore, Lys203 and Glu287 have been identified as catalytic residues in a homologous protein. Based on the spatial positioning of these residues relative to the donor substrate, we proposed a front-face SNi reaction mechanism for GumH. To draw definitive conclusions regarding GumH’s structure, dynamics, and functional residues, experimental validation of the hypotheses presented in this work will be necessary. Datafiles Overview GumH was simulated using four distinct starting structures: AlphaFold3 Model 0 (AF) RoseTTAFold Model 0 (RF) Boltz-1, where GumH was co-folded with the acceptor substrate (Boltz) AlphaFold3 Model 2, where GumH was co-folded with GDP (MS) The latter two structures (Boltz and MS) were further refined using Rosetta’s kinematic loop remodeling and relax applications, resulting in closed conformations with trans-prolines. Code Used to Conduct Analyzes All Python code used for molecular dynamics (MD) trajectory analysis, as well as for running ClustENMD and normal mode analysis (NMA), is provided in the script md_nma_clustenmd.ipynb. The following data files are included: Coordinate files (.pdb) and trajectory files (.xtc, .dcd) used and generated during this work, organized in directories named according to the starting structure. Docking input and output files: GumH’s acceptor and donor substrates were docked using Boltz-1 and GNINA. All associated input files and scripts used to run these programs are located in the docking/ directory. Simulation input files: The GROMACS run files, including .mdp parameter files, making it possible to produce new simulations based on the same method. Simulations are organized into two categories: Membrane/ – systems where GumH was embedded in a membrane. Free_in_solution/ – systems where the protein was simulated in aqueous solution. Due to differences in system setup and equilibration protocols, slightly different simulation parameters were used for each condition.