Opposites Attract: Escherichia coli Heptosyltransferase I Conformational Changes Induced by Interactions between the Substrate and Positively Charged Residues.
Joy M CoteCody J S HechtKaelan R PatelCarlos A Ramirez-MondragonYuk Y ShamErika A TaylorPublished in: Biochemistry (2020)
Gram-negative bacterial viability is greatly reduced by the disruption of heptose sugar addition during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane component. Heptosyltransferase I (HepI), a member of the GT-B structural subclass of glycosyltransferases, is therefore an essential enzyme for the biosynthesis of the LPS. The disruption of HepI also increases the susceptibility of bacteria to hydrophobic antibiotics, making HepI a potential target for drug development. In this work, the structural and dynamic properties of the catalytic cycle of HepI are explored. Previously, substrate-induced stabilization of HepI was observed and hypothesized to be assisted by interactions between the substrate and residues located on dynamic loops. Herein, positively charged amino acids were probed to identify binding partners of the negatively charged phosphates and carboxylates of Kdo2-lipid A and its analogues. Mutant enzymes were characterized to explore changes in enzymatic activities and protein stability. Molecular modeling of HepI in the presence and absence of ligands was then performed with the wild type and mutant enzyme to allow determination of the relative change in substrate binding affinity resulting from each mutation. Together, these studies suggest that multiple residues are involved in mediating substrate binding, and a lack of additivity of these effects illustrates the functional redundancy of these binding interactions. The redundancy of residues mediating conformational transitions in HepI illustrates the evolutionary importance of these structural rearrangements for catalysis. This work enhances the understanding of HepI's protein dynamics and mechanism and is a model for improving our understanding of glycosyltransferase enzymes.
Keyphrases
- amino acid
- wild type
- gram negative
- binding protein
- escherichia coli
- inflammatory response
- molecular dynamics simulations
- multidrug resistant
- dna binding
- molecular dynamics
- toll like receptor
- single molecule
- lps induced
- genome wide
- climate change
- dna methylation
- high resolution
- staphylococcus aureus
- high glucose
- hydrogen peroxide
- oxidative stress
- ionic liquid
- hepatitis c virus
- diabetic rats
- nitric oxide
- human immunodeficiency virus
- drug induced
- risk assessment
- stress induced