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Bacterial antibiotic resistance is a rapidly expanding problem in the world today.

Functionalization of the outer membrane of Gram-negative bacteria provides protection from

extracellular antimicrobials, and serves as an innate resistance mechanism. Lipopolysaccharides (LPS)

are a major cell-surface component of Gram-negative bacteria that contribute to protecting the

bacteriumfromextracellular threats. LPS is biosynthesized by the sequential addition of sugarmoieties

by a number of glycosyltransferases (GTs). Heptosyltransferases catalyze the addition of multiple

heptose sugars to form the core region of LPS; there are at most four heptosyltransferases found in

all Gram-negative bacteria. The most studied of the four is HepI. Cells deficient …

Heptosyltransferase I (HepI) catalyzes the addition of L-glycero-β-Dmanno-heptose to 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 the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially ratelimiting, 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 …