Investigation of E. coli Heptosyltransferase I Dynamics

Interest in new methods to treat gram-negative bacterial infections has emerged due to a significant increase in antibiotic resistance amongst bacteria. Bacterial biofilms are a major contributor to this immunity. Heptosyltransferase-I (HepI) is an essential enzyme for the biosynthesis of lipopolysaccharides (LPS), an important component to bacterial biofilms. Cells deficient in HepI have decreased intestinal colonization and are more susceptible to hydrophobic antibacterials, which makes HepI a good target for developing inhibitors. HepI is a member of the GT-B structural subclass of glycosyltransferases. Crystal structures of GT-B enzymes have been observed to interconvert between open and closed conformations based up the ligation state of the proteins; we therefore hypothesize that HepI will also interconvert between open and closed conformations to enable catalysis. In HepI, there are eight tryptophan residues, which enable us to observe changes in the intrinsic tryptophan fluorescence upon substrate binding. Using wild-type and mutant forms of HepI we are attempting to discern which regions are undergoing conformational changes upon binding of the sugar acceptor substrate (associated with an observed blue shift in the fluorescence). Individual HepI tryptophan residues have been mutated to phenylalanine. Arginine residues that we hypothesize to have an important role in substrate induced conformational changes have also been mutated. Fluorescence circular dichroism have been used to determine the impact of these residues upon binding. Enzyme kinetics were also performed on all mutants to ensure that the mutagenesis was not impacting catalysis. Data thus far suggest that a conformational change is indeed needed for chemistry to occur. Monitoring whether a large dynamic closing occurs, is also being explored using mutagenesis and site specific fluorophore incorporation. Ultimately, an enhanced understanding of HepI's protein dynamics and mechanism may lead to the design of more effective gram-negative therapeutics.