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Erika A. Taylor




The virulence and/or viability of gram negative bacteria is greatly reduced when addition of a single -L-glycero-D-manno-heptose sugar (heptose) is prevented during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane (OM) component. Glycosyltransferase (GT) enzyme Heptosyltransferase I (HepI) mediates the nucleophilic addition of the first heptose onto Kdo2-lipid A. Cells deficient in HepI have decreased intestinal colonization and are more susceptible to hydrophobic antibacterials, which makes HepI a good target for developing inhibitors. The aim of this work is to decipher the structural and dynamic processes that are key to the catalytic cycle of HepI. HepI has two αβα Rossman-like substrate binding domains connected by an alpha helical linker region and characterized in the GT-B structural subclass of glycosyltransferases. Like other GT-B's, HepI is thought to interconvert between open and closed conformations to enable catalysis. A better understanding of these precesses will aid in development of dynamic inhibitors, of not only HepI but also other GT-B enzymes.

HepI contains eight tryptophan residues, which, through changes in the intrinsic tryptophan fluorescence, report upon substrate binding. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence individual HepI tryptophan residues were mutated to phenylalanine and the fluorescence data each mutant was compared to that for WT-HepI, allowing determination of regions of conformational dynamics. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose [ADPH]) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADPH binding site (with Trp217 in van der Waals contact with the adenine ring of ADPH), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI -heating to 95°C fails to denature the protein when HepIoODLA complex is formed.

To further decipher the origin of the HepIoODLA complex stabilization, ODLA binding, and ultimately HepI dynamics studies were performed with Lys and Arg mutants. Positively charged residues that we hypothesize to have an important role in ODLA binding were mutated to alanine. Circular dichroism experiments on all of the mutants and wild type, in conjunction with kinetic analyses were used to further examine the impact of these mutations. Fluorescence experiments allowed for testing the mutants potential role in substrate binding induced conformational changes. Data thus far suggest that multiple conformational changes are needed for chemistry to occur. Specifically, mutants W62F, W66F and W116F all displayed reductions in the observed blue shift upon ODLA binding, this suggests the that loops (N-3 and N-7) in which these residues are located become buried upon substrate binding. Additionally, positively charged residues (R63, K64 and R120), which are also located on the N-3 and N-7 loops, were identified to play an important role in substrate recognition and stability.

Ultimately, an enhanced understanding of HepI's protein dynamics and mechanism is expected to lead to the design of more effective antibacterial agents. These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and provide a toolbox of techniques that can be utilized to asses if dynamic inhibitors perturb conformational changes. Disruption of these dynamics may serve as a novel mechanism for inhibiting HepI and other glycosyltransferase enzymes.

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