The gastrointestinal disease cryptosporidiosis, caused by the genus Cryptosporidium, is a common cause of diarrheal diseases in children, particularly in developing countries and frequently fatal in immunocompromised individuals. Cryptosporidium hominis (Ch)-specific bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) has been a molecular target for inhibitor design. (Note that this bifunctional enzyme has also been referred to as TS-DHFR in previous literature since the functional biochemical reaction first involves the conversion of methylene tetrahydrofolate to dihydrofolate at the TS site.) While nanomolar inhibitors of Ch DHFR-TS have been identified at the biochemical level, effective delivery of these compounds to achieve anticryptosporidial activity in cell culture and in vivo models of parasite infection remains a major challenge in developing new therapies. Previous studies, using a nanotherapy approach, have shown a promising Ch DHFR-TS inhibitor, 906, that can successfully target Cryptosporidium parasites in cell culture with nanomolar anticryptosporidial activity. This formulation utilized poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with 906 (NP-906) and conjugated with a Cryptosporidium monoclonal antibody (MAb) on the nanoparticle surface to specifically target the glycoprotein GP25-200 in excysting oocysts. However, a limitation for in vivo use is antibody susceptibility to gastric acidity. To address this gap, a prodrug diethyl ester form of 906 (MAb-NP-Prodrug) was synthesized that allowed higher compound loading in the MAb-coated PLGA nanoparticles. An oral formulation was prepared by loading lyophilized MAb-NP-Prodrug into gelatin capsules with an enteric coating for gastric stability. Proof-of-concept studies with this oral formulation demonstrated antiparasitic activity in a chronic mouse model of Cryptosporidium infection. Efficacy was observed after a low daily dose of 2 x 8 mg kg-1 for 5 days, when examined 6 and 20 days postinfection, offering a new avenue of drug delivery to be further explored.

Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region "crossover helix" to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the non-active site pocket.

Thymidylate synthase (TS), found in all organisms, is an essential enzyme responsible for the de novo synthesis of deoxythymidine monophosphate. The TS active sites of the protozoal parasite Cryptosporidium hominis and human are relatively conserved. Evaluation of antifolate compound 1 and its R-enantiomer 2 against both enzymes reveals divergent inhibitor selectivity and enzyme stereospecificity. To establish how C. hominis and human TS (ChTS and hTS) selectively discriminate 1 and 2, respectively, we determined crystal structures of ChTS complexed with 2 and hTS complexed with 1 or 2. Coupled with the previously determined structure of ChTS complexed with 1, we discuss a possible mechanism for enzyme stereospecificity and inhibitor selectivity.

Cryptosporidiosis is a human gastrointestinal disease caused by protozoans of the genus Cryptosporidium, which can be fatal in immunocompromised individuals. The essential enzyme, thymidylate synthase (TS), is responsible for de novo synthesis of deoxythymidine monophosphate. The TS active site is relatively conserved between Cryptosporidium and human enzymes. In previous work, we identified compound 1, (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl-l-glutamic acid), as a promising selective Cryptosporidium hominis TS (ChTS) inhibitor. In the present study, we explore the structure-activity relationship around 1 glutamate moiety by synthesizing and biochemically evaluating the inhibitory activity of analogues against ChTS and human TS (hTS). X-Ray crystal structures were obtained for compounds bound to both ChTS and hTS. We establish the importance of the 2-phenylacetic acid moiety methylene linker in optimally positioning compounds 23, 24, and 25 within the active site. Moreover, through the comparison of structural data for 5, 14, 15, and 23 bound in both ChTS and hTS identified that active site rigidity is a driving force in determining inhibitor selectivity.

HepI (heptosyltransferase I from E. coli) catalyzes the addition of heptose from ADP-β-L-glycero-D-manno-heptose (ADPH) onto Kdo 2-Lipid A, a crucial step in the formation of lipopolysaccharides (LPS) in Gram-negative bacteria. LPS is a major cell surface component that maintains the structural integrity of bacterial outer membrane, contributes to the formation of bacterial biofilms, and provides protection from extracellular hazards. Disruption of HepI has been shown to result in a truncated LPS, and resulting bacteria display increased susceptibility to hydrophobic antibiotics. Due to increased bacterial antibiotic resistance in the biomedical field, the development of inhibitors for HepI is important as a possible means to enhance the function of existing antibiotics. HepI has been shown to be highly dynamic; its two domains appear to alternate between an “open” and “closed” structure upon substrate binding. HepI kinetics suggests that protein dynamics are partially rate-limiting, making the disruption of conformational changes during catalysis of interest in ongoing inhibitor design efforts. However, the global, dynamic mechanism of HepI is largely unknown. Here, we probe HepI dynamics with pyrene excimer fluorescence and tryptophan-induced quenching (TIQ) methods. To date, a full library of HepI mutants have been developed for the two studies to allow visualization of a variety of conformational changes before and during catalysis. The fluorescence spectrum of the pyrene probe mutant CHR (C297S, H221C, R61C) bound to both substrates showed a broadening of peaks in the excimer range (as compared to that of the apo protein), suggesting that a faint excimer had formed – consistent with significant conformational change of the protein. This and other constructs will be used to explore if fluorescence changes are observed during the course of catalysis. Understanding the complex conformational changes that occur during catalysis is the first step towards designing potential inhibitors for HepI. Support or Funding Information The authors would like to thank the National Institute of Health (R15AI119907) and the American Society of Biochemistry and Molecular Biology Undergraduate Research Award for funding this project. Locations and interactions of pyrene excimer and tryptophan-induced quenching molecular probes. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.

Heptosyltransferase I (HepI) catalyzes the addition of l-glycero-β-d-manno-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 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 changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA 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 it is 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 conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.

Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

Heptosyltransferase I (HepI), the enzyme responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide, 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 of a GT-B enzyme undergoing a substrate binding-induced transition from an open to closed state prior to catalysis.

Heptosyltransferase (Hep) enzymes are essential for the formation of bacterial biofilms in Gram negative bacteria, making the Hep enzymes an important target for the development of biofilm inhibitors. Our research is directed toward the detailed characterization of both the chemical mechanism of HepI, as well as the protein dynamics. We have already demonstrated the ability of this enzyme to accept a variety of simplified substrate analogues with catalytic efficiencies as good or better than with the native substrate. GTs of the GT-B structural fold, like HepI, are characterized by having two beta/alpha/beta rossman-like domains connected by a linker region, requiring a conformational change from open to closed states in order for catalysis to occur. Currently our lab is exploring these conformational changes through intrinsic tryptophan fluorescence. Using stopped-flow and steady state fluorescence analysis, in conjunction with mutagenisis, we are attempting to elucidate conformational changes that occur during the catalytic cycle. By combining analyses of the chemical mechanism with an understanding of protein dynamics in this system, this work is anticipated to lead to enhancements in drug discovery for this and other GT enzymes.

The increasing incidence of antibiotic resistant bacterial infections has necessitated the search for novel targets and mechanisms for therapeutic intervention. Formation of a biofilm enables survival of bacteria in hostile environments, including in the presence of antimicrobials, thereby prompting research toward the disruption of bacterial biofilms. Lipopolysaccharide (LPS), a major constituent of the outer membrane of gram-negative bacteria, plays a key role in the formation and stability of biofilms. Therefore, disrupting the biosynthesis of the LPS is an attractive mechanism for development of anti-biofilm agents. One such LPS biosynthetic enzyme is heptosyltransferase I (Hep I), which is responsible for the transfer of the first L-glycero-D-manno-heptose to a 3-deoxy-α-D-manno-oct-2-ulopyranosonic acid (KDO) of the growing Lipid A portion of LPS. Previous attempts to co-crystallize Hep I with both substrates have been unsuccessful, hindering the ability to effectively visualize the binding interactions important for specificity. Substrate analogues for E. coli Hep I Lipid A are being used to identify which portions are necessary and sufficient for binding. Determination of the SAR of Hep I for these analogues may lead to the discovery of a Lipid A analogue that is able to be co-crystallized with Hep I. Developing an understanding of E.coli Hep I binding interactions with lipid A will enhance efforts to develop a Hep I inhibitor and possibly the discovery of a new anti-biofilm agent.

OBJECTIVE Impaired glucose counterregulation during hypoglycemia is well documented in patients with type 1 diabetes; however, the molecular mechanisms underlying this defect remain uncertain. We reported that the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in a crucial glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), plays an important role in modulating the magnitude of the glucagon and epinephrine responses to hypoglycemia and investigated whether VMH GABAergic tone is altered in diabetes and therefore might contribute to defective counterregulatory responses. RESEARCH DESIGN AND METHODS We used immunoblots to measure GAD65 protein (a rate-limiting enzyme in GABA synthesis) and microdialysis to measure extracellular GABA levels in the VMH of two diabetic rat models, the diabetic BB rat and the streptozotocin (STZ)-induced diabetic rat, and compared them with nondiabetic controls. RESULTS Both diabetic rat models exhibited an ~50% increase in GAD65 protein as well as a twofold increase in VMH GABA levels compared with controls under baseline conditions. Moreover, during hypoglycemia, VMH GABA levels did not change in the diabetic animals, whereas they significantly declined in nondiabetic animals. As expected, glucagon responses were absent and epinephrine responses were attenuated in diabetic rats compared with their nondiabetic control counterparts. The defective counterregulatory response in STZ-diabetic animals was restored to normal with either local blockade of GABAA receptors or knockdown of GAD65 in the VMH. CONCLUSIONS These data suggest that increased VMH GABAergic inhibition is an important contributor to the absent glucagon response to hypoglycemia and the development of counterregulatory failure in type 1 diabetes.

Heptosyltransferase I (HepI) is 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). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k(cat)/K(m) of 2.7 × 10(6) M(-1) s(-1), compared to a value of 2.2 × 10(5) M(-1) s(-1) for the Kdo(2)-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.

Heptosyltransferase I (HepI) is responsible for the transfer of L-glycero-D-manno-heptose to a 3-deoxyα-D-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a kcat/Km of 2.7 × 10 6 M−1 s−1, compared to a value of 2.2 × 10 M−1 s−1 for the Kdo2LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates. T effectiveness of antibiotics for the treatment of Gramnegative infections is hampered by their uptake into cells, because of the presence of an outer membrane containing a high level of lipopolysaccharide (LPS). LPS on cell surfaces is important for cell motility, intestinal colonization and bacterial biofilm formation, and contributes substantively to antibiotic resistance. These characteristics have spurred research toward the development of inhibitors for the LPS biosynthetic enzymes. The structure of the LPS is comprised of three parts: LipidA, core oligosaccharide (Core-OS), and repeating O-Antigen. Heptosyltransferase I (HepI) catalyzes the first step in the LPS synthesis pathway following lipid functionalization of LipidA. Blocking LPS biosynthesis prior to the addition of an L-glycero-D-manno-heptose (Hep) residue results in increased bacterial sensitivity to hydrophobic antibiotics and phagocytosis by macrophages; thus, HepI is considered an excellent target for inhibitor development. In our efforts to determine the enzyme mechanism for drug design, we have found that HepI can efficiently utilize multiple LipidA analogues as substrates, including a completely lipid free LipidA molecule. This is the first enzyme in the LPS biosynthetic pathway that does not have strict selectivity for acylated substrates. Furthermore, this finding has important implications for the substrate selectivity of downstream enzymes, which are now thought also to utilize deacylated substrates. HepI is essential for the transfer of the first Hep moiety in the core oligosaccharide of LPS. It catalyzes formation of an α(1→5) linkage between L-glycero-D-manno-heptose and the first 3-deoxy-α-D-oct-2-ulopyranosonic acid (Kdo) covalently attached to LipidA (Scheme 1). A crystal structure of Escherichia coli HepI bound to a donor substrate analogue, ADP-2-deoxy-2-fluoroheptose, reveals binding interactions between ADP-Hep and the sugar donor domain; however, these structures revealed little about interactions of HepI with the acceptor substrate Kdo2-LipidA. 6 Our goal was to identify a substrate analogue that would be amenable to crystallography, thereby allowing the residues that confer sugar acceptor specificity to be revealed, while also attempting to identify tighter binding molecules that lead toward the development of HepI inhibitors. Deacylation of the LipidA molecules was investigated to improve the solubility of the acceptor substrate, thereby potentially removing the need for detergent, while also removing potential entropic penalties associated with binding. Additionally, because the Kdo2-LipidA molecule is believed to be bound in the membrane during catalysis, we hypothesized that these residues would be relatively unimportant for conferring specificity. Here we report the effect of the acylation state, as well as the number of Kdo molecules, has on Km for the acceptor substrate. Kdo2-LipidA (Figure 1A) was isolated from E. coli HepI knockout strain WBB06 according to published protocols, and Kdo-LipidA (Figure 1B, also known as Re-LipidA) was obtained from Sigma-Aldrich. Kdo2-LipidA was O-deacylated by reaction with anhydrous hydrazine. The fully deacylated acceptor molecule (Figure 1D), which was previously synthesized by Brabetz and coworkers, was synthesized using a modified method analogous to that used by Bystrova and coworkers. First, the O-deacylated Kdo2LipidA (Figure 1C) was made via reaction with anhydrous hydrazine and then the fully deacylated acceptor resulted from subsequent refluxing with 4 M NaOH. Using a coupled assay containing pyruvate kinase and L-lactate dehydrogenase, HepI activity was determined through UV−vis monitoring of production of nicotinamide adenine dinucleotide at 340 nm. All four LipidA analogues (Figure 1A−D) were found to be competent substrates when the assay was conducted with or without Triton X-100 (Table 1). Formation of the pentasaccharide (Scheme 1) derived from use of the fully deacylated LipidA analogue (Figure 1D) was confirmed by ESI-MS (see the Supporting Information). A roughly 5-fold decrease in the rate of turnover was observed between the fully acylated acceptor substrates and the deacylated forms of Kdo2-LipidA, with similar rates observed for both the O-deacylated and fully deacylated LipidA analogues. This decrease in kcat is more than offset by an increased binding affinity, causing the overall catalytic efficiency to be 4-fold greater for Received: October 14, 2011 Revised: November 6, 2011 Published: November 7, 2011 Rapid Report pubs.acs.org/biochemistry © 2011 American Chemical Society 10570 dx.doi.org/10.1021/bi201581b |Biochemistry 2011, 50, 10570−10572 the fully deacylated Kdo2-LipidA than for the fully acylated LipidA (kcat/Km values of 2.1 × 10 6 and 5.2 × 10 M−1 s−1, respectively) in the presence of Triton X-100 and 12-fold greater (kcat/Km values of 2.7 × 10 6 and 2.2 × 10 M−1 s−1, respectively) in the absence of detergent. Furthermore, the <2-fold binding affinity discrimination between Kdo-LipidA and Kdo2-LipidA can be rationalized because in vivo it has been demonstrated that the E. coli Kdo transferase (EcKdtA) is a bifunctional enzyme that catalyzes incorporation of both Kdo units. Because a single incorporation of Kdo is only observed using isolated EcKdtA with limiting quantities of its sugar donor, cytosine monophosphate-Kdo, HepI would only encounter Kdo2-LipidA in vivo, thereby eliminating any driving force for selectivity between Kdo-LipidA and Kdo2-LipidA. Tighter binding of the fully deacylated acceptor analogue, when compared to the acylated acceptors, is enhanced further in the absence of detergent. We hypothesize that when the acyl chains are contained within a detergent micelle, they are isolated from the enzyme, but when free in solution (in the absence of detergent), they make destabilizing interactions with the protein surface or aggregate together. This hypothesis is supported by the observation that HepI aggregates in the presence of Triton X-100, the fully acylated Kdo2-LipidA, and the combination, whereas apo HepI and HepI bound to the fully deacylated LipidA do not show significant aggregation when analyzed by size exclusion chromatography (data not Scheme 1. Reaction Catalyzed by E. coli HepI Figure 1. Structures of LipidA acceptor substrates: (A) E. coli Kdo2LipidA, (B) Salmonella minnesota Kdo-LipidA, (C) O-deacylated E. coli Kdo2-LipidA, and (D) fully deacylated E. coli Kdo2-LipidA. The Kdo that acts as a nucleophile for the glysyltransferase reaction is colored red. Biochemistry Rapid Report dx.doi.org/10.1021/bi201581b |Biochemistry 2011, 50, 10570−10572 10571 shown). Furthermore, the observation that the fully deacylated acceptor has a higher affinity in the absence of detergent, and the opposite is true for the O-deacylated LipidA analogue, provides further support for our hypothesis. Because the acyl portion of the LipidA acceptor resides in the inner leaflet of the inner membrane when the reaction occurs in vivo, we speculate that the enzyme only recognizes the sugar region of these acceptor molecules for catalysis. This is the first report of enzymatic activity for HepI in the absence of detergent, suggesting that perhaps the sensitivity of the coupled assay for determining initial rates is also an improvement over traditional thin layer chromatography analysis of radioisotope incorporation for LPS elongation. The small differences in kinetic parameters among these four substrates suggest that the sugar binding interactions of the acceptor substrate predominate for HepI. Our findings are consistent with a prior report that Kdo2-LipidIVA (a tetraactylated LipidA analogue that is biosynthetically upstream of HepI) can be adventitiously turned over by HepI and has an apparent Km of 4.5 μM. 11 The ability of HepI not only to utilize an underacylated LipidA analogue but also to have improved catalytic efficiency in the absence of any lipids has broad implications for enzymes that utilize glycolipid substrates, and especially other enzymes of the LPS biosynthetic pathway. Furthermore, our finding that fully deacylated Kdo2-LipidA is a better substrate for the enzyme than the native substrate, and that it binds without inducing aggregation, should open up new strategies for structural analysis of the protein−substrate interactions of this and related glycolipid-modifying enzymes. The ability of this enzyme to utilize multiple LipidA analogues suggests that examination of the glycosyltransferases involved in LPS biosynthesis, including HepI, could yield answers that are broadly applicable to questions concerning substrate selectivity in glycosyltransferases in general. We anticipate that these results will ultimately enhance lead discovery in the area of inhibition of LPS biosynthesis for treatment of Gram-negative bacterial infections. ■ ASSOCIATED CONTENT *S Supporting Information LPS deacylation procedure, HepI purification, detailed assay conditions, and ESI-MS data from reaction of the fully deacylated Kdo2-LipidA substrate with HepI. This material is available free of charge via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *Department of Chemistry, Wesleyan University, Middletown, CT 06459. P