The virulence and/or viability of gram negative bacteria is greatly reduced when addition of a single L-glycero-β-D-manno-heptose sugar (heptose) is prevented during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane (OM) component. The 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, which is a member of the GT-B structural subclass of glycosyltransferases. Like other GT-B’s, HepI has two two Rossman-like substrate binding domains connected by an alpha helical linker region. HepI is thought to interconvert between open and closed conformations to enable catalysis. In HepI, there are eight tryptophan residues, which, through changes in the intrinsic tryptophan fluorescence, report upon substrate binding. Using wild-type and mutant forms of HepI we are attempting to discern which regions and on what timescales conformational changes are occurring (associated with an observed blue shift in the fluorescence) during catalysis. Individual HepI tryptophan residues have been mutated to phenylalanine and the fluorescence data for the mutants is compared to that for WT, allowing determination of regions of conformational dynamics. In addition, arginine residues that we hypothesize to have an important role in substrate binding induced conformational changes were mutated to alanine. Fluorescence experiments are allowing us to test their potential role in substrate binding induced conformational changes. Circular dichroism experiments on all of the mutants and wild type, in conjunction with kinetic analyses are used to further examine the impact of these mutations. Data thus far suggest that multiple conformational changes are needed for chemistry to occur. Ultimately, an enhanced understanding of HepI’s protein dynamics and mechanism is expected to lead to the design of more effective antibacterial agents.

Using the LPS biosynthetic enzyme heptosyltansferase I to explore protein dynamics of a GT-B glycosyltrasferase

253rd National Meeting (2017)

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