Heptosyltransferase I (HepI) catalyzes the addition of L-glycero-β-D-manno-heptose onto Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making design of inhibitors against HepI of interest. Since HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via monitoring fluorescence wavelength changes. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-Heptose) are in a more hydrophobic environment upon ODLA binding to the N-terminal domain. These residues are adjacent to the ADP-Heptose binding site (with Trp217 in van der Waals contact with the Adenine ring of ADP-Heptose), 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 in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these dynamics may serve as novel mechanism for inhibiting this and other glycosyltransferase enzymes.

Heptosyltransferase I (HepI), the enzyme responsible for the transfer of L-glycero-D-manno-heptose to a 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS), is a member of the GT-B structural class of enzymes. Crystal structures have revealed open and closed conformations of apo and ligand-bound GT-B enzymes, implying that large-scale protein conformational dynamics play a role in their reaction mechanism. Here we report transient kinetic analysis of conformational changes in HepI reported by intrinsic tryptophan fluorescence, and present the first real time evidence for a GT-B enzyme undergoing substrate binding-induced transition from an open to closed state prior to catalysis.

Class D β-lactamases, a major source of bacterial resistance to β-lactam antibiotic therapies, represent a distinct subset of the β-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multiple sequence alignment and phylogenetic tree of ∼80 Class D β-lactamases that leverage several 3D structures of these enzymes. We observe several sequence clusters on the phylogenetic tree, some that are species specific while others include several species from α-, β- and γ-proteobacteria. Residues characteristic of a specific cluster were identified and shown to be located just outside the active site, possibly modulating the function of the catalytic residues to facilitate reactions with specific types of β-lactams. Most significant was the discovery of a likely disulfide bond in a large group composed of α-, β- and γ-proteobacteria that would contribute to enzyme stability and hence bacterial viability under antibiotic assault. A network of co-evolving residues was identified which suggested the importance of maintaining a surface for binding a highly conserved Phe69.

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 Kdo 2 -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.