Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Structure of the metal-dependent deacetylase LpxC from Yersinia enterocolitica complexed with the potent inhibitor CHIR-090

The first committed step of lipid A biosynthesis is catalyzed by UDP-(3-O-((R)-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase, a metal-dependent deacetylase also known as LpxC. Because lipid A is essential for bacterial viability, the inhibition of LpxC is an appealing therapeutic strategy for the treatment of Gram-negative bacterial infections. Here we report the 1.79 ? resolution X-ray crystal structure of LpxC from Yersinia enterocolitica (YeLpxC) complexed with the potent hydroxamate inhibitor CHIR-090. This enzyme is a nearly identical orthologue of LpxC from Yersinia pestis (99.7% sequence identity), the pathogen that causes bubonic plague. Similar to the inhibition of LpxC from Escherichia coli, CHIR-090 inhibits YeLpxC via a two-step slow, tight-binding mechanism with an apparent K(i) of 0.54 ± 0.14 nM followed by conversion of the E·I to E·I* species with a rate constant of 0.11 ± 0.01 min(-1). The structure of the LpxC complex with CHIR-090 shows that the inhibitor hydroxamate group chelates the active site zinc ion, and the "tail" of the inhibitor binds in the hydrophobic tunnel in the active site. This hydrophobic tunnel is framed by a βαβ subdomain that exhibits significant conformational flexibility as it accommodates inhibitor binding. CHIR-090 displays a 27 mm zone of inhibition against Y. enterocolitica in a Kirby-Bauer antibiotic assay, which is comparable to its reported activity against other Gram-negative species including E. coli and Pseudomonas aeruginosa. This study demonstrates that the inhibition of LpxC should be explored as a potential therapeutic and/or prophylatic response to infection by weaponized Yersinia species.     

Molecular recognition by Escherichia coli UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase is modulated by bound metal ions

The metal-dependent enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the conversion of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)glucosamine and acetate. This is the committed step in the biosynthesis of lipid A, and for this reason, LpxC is a target for the development of antibiotics in the treatment of Gram-negative bacterial infections. Here we examine the importance of bound metal ion(s) and fatty acids for molecular recognition of ligands by LpxC. The KDproduct value increases >1000-fold with the loss of the hydroxymyristoyl moiety, indicating that the enhanced catalytic efficiency of substrates containing this acyl group is mainly due to increased binding affinity. New fluorescent binding assays for measuring the affinity of LpxC for fatty acids indicate that myristate binds to LpxC 10-fold less tightly than palmitate and that fatty acid affinity is only modestly dependent on pH. Furthermore, LpxC homologues from different species have similar affinities for fatty acids despite alterations in protein sequence. In contrast, the affinity of LpxC for both product and fatty acids is significantly influenced (< or =40-fold) by changes in the number and identity of metal ions bound to the LpxC active site. Therefore, interactions with these metal ions are critical for molecular recognition of ligands by LpxC and may mimic similar contacts with active site inhibitors. These data indicate that the potency of LpxC inhibitors in vitro can be altered by assay conditions used in screening and/or development of LpxC inhibitors and that the metal ion status of LpxC in vivo will likely influence the effectiveness of LpxC inhibitors as antibiotics.     

Crystal structure of A. aeolicus LpxC with bound product suggests alternate deacetylation mechanism

UDP‐3‐O‐acyl‐N‐acetylglucosamine deacetylase (LpxC) is the first committed step to form lipid A, an essential component of the outer membrane of Gram‐negative bacteria. As it is essential for the survival of many pathogens, LpxC is an attractive target for antibacterial therapeutics. Herein, we report the product‐bound co‐crystal structure of LpxC from the acheal Aquifex aeolicus solved to 1.6 Å resolution. We identified interactions by hydroxyl and hydroxymethyl substituents of the product glucosamine ring that may enable new insights to exploit waters in the active site for structure‐based design of LpxC inhibitors with novel scaffolds. By using this product structure, we have performed quantum mechanical modeling on the substrate in the active site. Based on our results and published experimental data, we propose a new mechanism that may lead to a better understanding of LpxC catalysis and inhibition. Proteins 2015; 83:1706–1719. © 2015 Wiley Periodicals, Inc.     

Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC). Identification of the zinc binding site

UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria. Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli. A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles. Each amino acid was altered in both E. coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined. Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions. H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+). The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC. The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion. Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics.     

Structure of the LpxC deacetylase with a bound substrate-analog inhibitor

The zinc-dependent UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) that constitutes the outermost monolayer of Gram-negative bacteria. As LpxC is crucial for the survival of Gram-negative organisms and has no sequence homology to known mammalian deacetylases or amidases, it is an excellent target for the design of new antibiotics. The solution structure of LpxC from Aquifex aeolicus in complex with a substrate-analog inhibitor, TU-514, reveals a novel alpha/beta fold, a unique zinc-binding motif and a hydrophobic passage that captures the acyl chain of the inhibitor. On the basis of biochemical and structural studies, we propose a catalytic mechanism for LpxC, suggest a model for substrate binding and provide evidence that mobility and dynamics in structural motifs close to the active site have key roles in the capture of the substrate.     

EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC)

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.     

Catalytic mechanism and molecular recognition of E. coli UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase probed by mutagenesis

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metal-dependent deacetylase that catalyzes the hydrolysis of UDP-3-O-myristoyl-N-acetyl-glucosamine to form UDP-3-O-myristoyl-glucosamine and acetate. This is the committed step in the biosynthesis of lipid A, and therefore, LpxC is a target for the development of antimicrobial agents in the treatment of Gram-negative infections. To facilitate the development of potent and specific inhibitors of LpxC, the molecular determinants of binding and specificity and the catalytic mechanism for this enzyme have been probed. The functions of active site residues have been classified on the basis of changes in steady-state turnover (kcat, KM, and kcat/KM) and product binding affinity (KDProduct). We have identified side chains that enhance product affinity and reactivity (F192, K239, D246, and H265), destabilize product affinity (E78 and D197), and preferentially enhance catalytic efficiency (H19, T19, K143, and N162). In addition, the affinity of LpxC for myrUDP-GlcNH2 is dependent on two ionizations, one deprotonation and one protonation, with apparent pKa values of 6.5 +/- 0.1 and 7.4 +/- 0.1, respectively. The UDP moiety of the product contributes significantly to recognition by LpxC, suggesting that this region can be targeted in drug development. These data provide a map of the active site features essential for catalysis and molecular recognition by LpxC that can be used for developing more potent LpxC inhibitors.     

Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition, and binding mode study of SAHA-based non-hydroxamates

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i) substrate (acetyl lysine) analogues (compounds 3–7), (ii) analogues bearing various functional groups expected to chelate zinc ion (compounds 8–15), and (iii) analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16–18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.     

Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.     

Crystal structure of LpxC from Pseudomonas aeruginosa complexed with the potent BB-78485 inhibitor

The cell wall in Gram-negative bacteria is surrounded by an outer membrane comprised of charged lipopolysaccharide (LPS) molecules that prevent entry of hydrophobic agents into the cell and protect the bacterium from many antibiotics. The hydrophobic anchor of LPS is lipid A, the biosynthesis of which is essential for bacterial growth and viability. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is an essential zinc-dependant enzyme that catalyzes the conversion of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)glucosamine and acetate in the biosynthesis of lipid A, and for this reason, LpxC is an attractive target for antibacterial drug discovery. Here we disclose a 1.9 A resolution crystal structure of LpxC from Pseudomonas aeruginosa (paLpxC) in a complex with the potent BB-78485 inhibitor. To our knowledge, this is the first crystal structure of LpxC with a small-molecule inhibitor that shows antibacterial activity against a wide range of Gram-negative pathogens. Accordingly, this structure can provide important information for lead optimization and rational design of the effective small-molecule LpxC inhibitors for successful treatment of Gram-negative infections.     

Docking and binding mode analysis of aryl diketoacids (ADK) at the active site of HCV RNA-dependent RNA polymerase

The pharmacophore-guided docking study of aryl diketoacid (ADK) analogues revealed two distinctive hydrophobic binding sites (a pocket and a groove) around the UTP-binding site of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). Interestingly, the hydrophobic binding sites have appropriate shape and size to specifically substituted aromatic rings, which suggests the specific role of substituents on the aromatic ring in determining the binding affinity of the ADK analogue to the active site of the target enzyme. Binding mode analysis of ADK analogues with potent antiviral activity shows highly substituted aromatic rings map well onto the hydrophobic binding sites. For less active compounds, their lack of aromatic substitution and thereby insufficient size can be primarily ascribed to their inability to bind to the hydrophobic binding site. The characteristic binding mode of ADK analogues proposed in this study provides a useful tool in designing a structure–activity relationship study of novel ADK analogues based on various aromatic substituents.     

Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site.

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.     

Carbonic anhydrase inhibitors, interaction of boron derivatives with isozymes I and II: a new binding site for hydrophobic inhibitors at the entrance of the active site as shown by docking studies

The interaction of human carbonic anhydrase (hCA) isozymes I and II with boron derivatives was investigated by kinetic and spectroscopic studies. These derivatives, tested as new inhibitors of carbonic anhydrase, are sulfonamide and non-sulfonamide boron derivatives and some of them proved to be moderately efficient inhibitors of hCA I and hCA II, their activities being comparable to those of the unsubstituted sulfonamides, the classical inhibitors of these zinc enzymes. Ph(2) BOH, one of the compounds with the highest affinity for hCA II in the present study, has been docked within the active site. After minimisation it was found situated at 7.9 A from zinc, within the hydrophobic half of the active site, in Van der Waals contacts with the amino acid residues: Val 121, Phe 130, Val 135, Leu 141, Val 143, Val 207 and Pro 201. This is the first time that a CA inhibitor has been found to bind at the edge of the active site cavity, similarly to the CA activator histamine, which binds on the hydrophilic half. This finding may be of importance also for the design of novel types of inhibitors with increased affinity for the different CA isozymes.     

Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram-negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazoline-based hydroxamate that inhibits E. coli LpxC with K(i) of approximately 50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate-containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds inhibit both E. coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins."     

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase functions through a general acid-base catalyst pair mechanism

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of LpxC to metalloproteases led to the proposal that LpxC functions via a metalloprotease-like mechanism. The pH dependence of k(cat)/Km catalyzed by Escherichia coli and Aquifex aeolicus LpxC displayed a bell-shaped curve (EcLpxC yields apparent pKa values of 6.4+/-0.1 and 9.1+/-0.1), demonstrating that at least two ionizations are important for maximal activity. Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile is because of deprotonation of a zinc-coordinated group such as the zinc-water molecule, whereas the acidic limb of the pH profile is caused by protonation of either Glu78 or His265. Furthermore, the magnitude of the activity decreases and synergy observed for the active site mutants suggest that Glu78 and His265 act as a general acid-base catalyst pair. Crystal structures of LpxC complexed with cacodylate or palmitate demonstrate that both Glu78 and His265 hydrogen-bond with the same oxygen atom of the tetrahedral intermediate and the product carboxylate. These structural features suggest that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile.     

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity.     

Exploring the UDP pocket of LpxC through amino acid analogs

Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.     

Refined solution structure of the LpxC-TU-514 complex and pKa analysis of an active site histidine: insights into the mechanism and inhibitor design

Lipopolysaccharide, the major constituent of the outer monolayer of the outer membrane of Gram-negative bacteria, is anchored into the membrane through the hydrophobic moiety lipid A, a hexaacylated disaccharide. The zinc-dependent metalloamidase UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the second and committed step in the biosynthesis of lipid A. LpxC shows no homology to mammalian metalloamidases and is essential for cell viability, making it an important target for the development of novel antibacterial compounds. Recent NMR and X-ray studies of the LpxC from Aquifex aeolicus have provided the first structural information about this family of proteins. Insight into the catalytic mechanism and the design of effective inhibitors could be facilitated by more detailed structural and biochemical studies that define substrate-protein interactions and the roles of specific residues in the active site. Here, we report the synthesis of the (13)C-labeled substrate-analogue inhibitor TU-514, and the subsequent refinement of the solution structure of the A. aeolicus LpxC-TU-514 complex using residual dipolar couplings. We also reevaluate the catalytic role of an active site histidine, H253, on the basis of both its pK(a) as determined by NMR titration and pH-dependent kinetic analyses. These results provide a structural basis for the design of more potent LpxC inhibitors than those that are currently available.