Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase

Glycosyltransferase (GT) enzymes promote the formation of glycosidic bonds between a sugar molecule and a diversity of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-n-manno-heptose, Hep) onto a lipid-anchored glycopolymer (heptosylated Kdo,Lipid A, Hep-Kdo(2)-Lipid A, or HLA). LPS plays a key role in Gram-negative bacterial sepsis, biofilm formation, and host colonization, and as such, LPS biosynthetic enzymes are targets for novel antimicrobial therapeutics. Three heptosyltransferases are involved in the inner-core LPS biosynthesis, with Escherichia coli HepII being the last to be quantitatively characterized in vivo. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here, we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared with HepI, including sugar donor promiscuity and sugar acceptor-induced secondary structural changes, which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates compared to HepI. A structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe the potentially important substrate-protein contacts. Ligand binding-induced changes in Trp fluorescence in HepII enabled the determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent, and specific inhibitors for this family of enzymes.

Automated summary

This study provides the first kinetic and biophysical characterization of HepII, revealing its shared properties with HepI, such as donor promiscuity and acceptor-induced structural changes. HepII exhibits higher catalytic efficiency and stronger binding affinity for substrates compared to HepI.