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• W2000786667 abstract "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 kcat/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 (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 kcat/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. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; LpxC, UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase; myr-UDP-GlcNAc, UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine; GABC, general acid-general base catalyst; GBC, general base catalyst; AaLpxC, A. aeolicus LpxC; EcLpxC, E. coli LpxC: MES, 4-morpholineethanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CAPS, 3-(cyclohexylamino)propanesulfonic acid; WT, wild type. 1The abbreviations used are: LPS, lipopolysaccharide; LpxC, UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase; myr-UDP-GlcNAc, UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine; GABC, general acid-general base catalyst; GBC, general base catalyst; AaLpxC, A. aeolicus LpxC; EcLpxC, E. coli LpxC: MES, 4-morpholineethanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CAPS, 3-(cyclohexylamino)propanesulfonic acid; WT, wild type. molecules form the outer membrane of Gram-negative bacteria and serve to exclude hydrophobic and negatively charged molecules. Lipid A is the hydrophobic portion of LPS that is responsible for anchoring LPS to the membrane and is essential for the viability of Gram-negative bacteria (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar). Lipid A is also known as endotoxin and is the immunomodulatory portion of LPS that triggers the immune system in septic shock. As a consequence, inhibition of lipid A biosynthesis is proposed as a strategy for both the development of novel antibiotics and anti-endotoxins in the treatment of septic shock (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar, 2White R.J. Margolis P.S. Trias J. Yuan Z.Y. Curr. Opin. Pharmacol. 2003; 3: 502-507Crossref PubMed Scopus (47) Google Scholar, 3Onishi 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. Raetz C.R.H. Science. 1996; 274: 980-982Crossref PubMed Scopus (342) Google Scholar, 4Wyckoff T.J.O. Raetz C.R.H. Jackman J.E. Trends Microbiol. 1998; 6: 154-159Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 5Manocha S. Feinstein D. Kumar A. Exp. Opin. Investig. Drugs. 2002; 11: 1795-1812Crossref PubMed Scopus (47) Google Scholar). In Escherichia coli lipid A is synthesized from UDP-N-acetylglucosamine in a 10-step pathway (Fig. 1). UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the hydrolysis of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine (myr-UDP-GlcNAc) to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar). The deacetylation of myr-UDP-GlcNAc is the committed step in the biosynthesis of lipid A (7Anderson M. Bull H. Galloway S. Kelly T. Mohan S. Radika K. Raetz C. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar); therefore, this enzyme is a target for the development of inhibitors as antibiotics for the treatment of Gram-negative infections (2White R.J. Margolis P.S. Trias J. Yuan Z.Y. Curr. Opin. Pharmacol. 2003; 3: 502-507Crossref PubMed Scopus (47) Google Scholar, 8Jackman J.E. Fierke C.A. Tumey L.N. Pirrung M. Uchiyama T. Tahir S.H. Hindsgaul O. Raetz C.R.H. J. Biol. Chem. 2000; 275: 11002-11009Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9Pirrung M.C. Tumey L.N. Raetz C.R.H. Jackman J.E. Snehalatha K. McClerren A.L. Fierke C.A. Gantt S.L. Rusche K.M. J. Med. Chem. 2002; 45: 4359-4370Crossref PubMed Scopus (101) Google Scholar, 10Clements J.M. Coignard F. Johnson I. Chandler S. Palan S. Waller A. Wijkmans J. Hunter M.G. Antimicrob. Agents Chemother. 2002; 46: 1793-1799Crossref PubMed Scopus (163) Google Scholar). A comprehensive understanding of the catalytic mechanism and structure of LpxC will facilitate the development of potent and specific inhibitors of this enzyme. The enzyme LpxC belongs to a group of enzymes known as the zinc hydrolases. Mononuclear zinc hydrolases can be broadly categorized by two general catalytic mechanisms as follows: one that uses a single bifunctional general acid-general base catalyst (GABC) (i.e. metalloproteases), and a second mechanism that uses a GABC pair (i.e. histone deacetylases) to carry out catalysis (11Christianson D.W. Lipscomb W.N. Acc. Chem. Res. 1989; 22: 62-69Crossref Scopus (644) Google Scholar, 12Aoki S. Kimura E. Comp. Coord. Chem. II. 2004; 8: 601-640Google Scholar, 13Lipscomb W.N. Strater N. Chem. Rev. 1996; 96: 2375-2433Crossref PubMed Scopus (1269) Google Scholar, 14Hernick M. Fierke C.A. Arch. Biochem. Biophys. 2005; 433: 71-84Crossref PubMed Scopus (149) Google Scholar). The structure of Aquifex aeolicus LpxC (AaLpxC) has been solved using x-ray crystallography (15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar) and NMR spectroscopy (16Coggins B.E. Li X.C. McClerren A.L. Hindsgaul O. Raetz C.R.H. Zhou P. Nat. Struct. Biol. 2003; 10: 645-651Crossref PubMed Scopus (90) Google Scholar). This structure (Fig. 2) reveals that LpxC contains a unique fold and a novel zinc-binding motif, both of which distinguish LpxC from other known zinc hydrolases (15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar). One bound zinc ion is an essential cofactor for LpxC catalytic activity (ZnA), whereas excess zinc leads to enzyme inhibition (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar) by ZnB (Fig. 2b) (15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar). The catalytic zinc ion (ZnA) is coordinated by His79, His238, Asp242, and a solvent molecule, whereas the inhibitory ZnB ion is coordinated by Glu78, His265, a fatty acid, and a bridging solvent molecule. Previous mutagenesis experiments suggest that three active site residues (Glu78, Asp246, and His265) are important for catalytic activity, as mutation of these residues to Ala decreases the catalytic activity >102-fold (17Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 2001; 40: 514-523Crossref PubMed Scopus (45) Google Scholar). On the basis of these structural and mutagenesis data, LpxC was proposed to function via a metalloprotease-like mechanism using a single general acid-base catalytic side chain (Glu78) and a zinc-water nucleophile to catalyze deacetylation of its substrate (Fig. 3A), as described in detail for metalloproteases (11Christianson D.W. Lipscomb W.N. Acc. Chem. Res. 1989; 22: 62-69Crossref Scopus (644) Google Scholar, 12Aoki S. Kimura E. Comp. Coord. Chem. II. 2004; 8: 601-640Google Scholar, 13Lipscomb W.N. Strater N. Chem. Rev. 1996; 96: 2375-2433Crossref PubMed Scopus (1269) Google Scholar, 15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar). The ZnA ion and His265 provide stabilization of the transition states and oxyanion intermediate prior to protonation of the amine leaving group and collapse of the oxyanion intermediate. In contrast, the deacetylation mechanism for histone deacetylases has been proposed to use a pair of His side chains, one functioning as a general acid and the other as a general base to activate the zinc-water nucleophile (12Aoki S. Kimura E. Comp. Coord. Chem. II. 2004; 8: 601-640Google Scholar, 18Finnin M.S. Donigian J.R. Cohen A. Richon V.M. Rifkind R.A. Marks P.A. Breslow R. Pavletich N.P. Nature. 1999; 401: 188-193Crossref PubMed Scopus (1485) Google Scholar). The details of the LpxC catalytic mechanism have not yet been fully elucidated. The pH dependence of AaLpxC wild-type and mutants has been reported recently (19McClerren A.L. Zhou P. Guan Z. Raetz C.R.H. Rudolph J. Biochemistry. 2005; 44: 1106-1113Crossref PubMed Scopus (40) Google Scholar), suggesting that Glu78 functions as a general base.Fig. 3The proposed mechanisms for LpxC using either a single bifunctional GABC (A) or a GABC pair (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Here we investigate the catalytic mechanism of LpxC using metal substitution, mutagenesis, pH dependence, and x-ray crystallography. The kcat/Km for E. coli LpxC (EcLpxC) has a bell-shaped dependence on pH with two pKa values of ∼6.4 and 9.1 similar to values recently reported for AaLpxC (19McClerren A.L. Zhou P. Guan Z. Raetz C.R.H. Rudolph J. Biochemistry. 2005; 44: 1106-1113Crossref PubMed Scopus (40) Google Scholar). Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile represents ionization of a metal-coordinated group, such as the zinc-water molecule. Kinetic evaluation of LpxC mutants and crystal structures of LpxC complexed with cacodylate or palmitate support a mechanism in which Glu78 and His265 function as a general acid-base catalyst pair, wherein the His265 side chain functions as a general acid to protonate the amine leaving group, whereas Glu78 functions as a general base to activate the zinc-water nucleophile. Mutagenesis and Protein Expression—All mutant plasmids were prepared using the QuickChange site-directed mutagenesis kit (Stratagene). The LpxC variants were overexpressed and purified according to published procedures (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar, 8Jackman J.E. Fierke C.A. Tumey L.N. Pirrung M. Uchiyama T. Tahir S.H. Hindsgaul O. Raetz C.R.H. J. Biol. Chem. 2000; 275: 11002-11009Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 20Young K. Silver L.L. Bramhill D. Cameron P. Eveland S.S. Raetz C.R.H. Hyland S.A. Anderson M.S. J. Biol. Chem. 1995; 270: 30384-30391Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 21McClure C.P. Rusche K.M. Peariso K. Jackman J.E. Fierke C.A. Penner-Hahn J.E. J. Inorg. Biochem. 2003; 94: 78-85Crossref PubMed Scopus (34) Google Scholar). For expression of less stable LpxC mutants, cells were incubated overnight at 25 °C following addition of isopropyl d-thiogalactopyranoside. Additionally, 100 μm isopropyl d-thiogalactopyranoside and 200 μm ZnSO4 were added at induction for expression of the D246A EcLpxC mutant. All purification steps were carried out at room temperature and 4 °C for AaLpxC and EcLpxC, respectively. Following lysis using a microfluidizer, the cell debris was pelleted by centrifugation, and the supernatant was loaded onto a DEAE-Sepharose column in 25 mm HEPES, pH 7, 2 mm dithiothreitol. Both AaLpxC and EcLpxC were eluted with a linear gradient (0–250 mm KCl). The LpxC was further fractionated using a Reactive Red 120 column with a linear gradient (0–250 mm KCl for EcLpxC and 150–500 mm KCl for AaLpxC). The LpxC enzymes were >95% pure as assessed by SDS-PAGE and were stored at –80 °C. All metals were removed from the purified enzymes by incubation with 20 mm dipicolinic acid, as described previously (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar). The apoLpxC was reconstituted with stoichiometric amounts of ZnSO4, CoSO4, or NiSO4 prior to use in assays (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar). The final metal-enzyme stoichiometry was determined using inductively coupled plasma emission mass spectrometry. For LpxC prepared using these methods, no fatty acid contaminants were detected by mass spectrometry following extraction of LpxC with methylene chloride. Preparation of Substrate—[14C]UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine was prepared from [14C]UDP-GlcNAc (PerkinElmer Life Sciences) catalyzed by UDP-3-O-acyltransferase (LpxA) and purified according to procedures used for preparation of [32P]UDP-3-O-((R)-hydroxymyristoyl)-N-acetylglucosamine (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar, 22Kelly T.M. Stachula S.A. Raetz C.R.H. Anderson M.S. J. Biol. Chem. 1993; 268: 19866-19874Abstract Full Text PDF PubMed Google Scholar). The plasmid encoding UDP-3-O-acyltransferase was generously provided by C. R. H. Raetz, Duke University (7Anderson M. Bull H. Galloway S. Kelly T. Mohan S. Radika K. Raetz C. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar). The substrate was obtained in 90–99% yield, as determined by scintillation counting. TLC analysis verified that the purification procedures removed any contaminating UDP-GlcNAc. Additionally, >90% of the synthesized myr-UDP-GlcNAc was cleaved by LpxC. LpxC Assay—The deacetylase activity was measured as described previously (6Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (109) Google Scholar, 23Sorensen P.G. Lutkenhaus J. Young K. Eveland S.S. Anderson M.S. Raetz C.R.H. J. Biol. Chem. 1996; 271: 25898-25905Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Briefly, assay mixtures containing buffer, bovine serum albumin (fatty acid-free, 1 mg/ml), triscarboxyethylphosphine (0.5 mm), and [14C]UDP-3-O-((R)-hydroxymyristoyl)-N-acetylglucosamine were pre-equilibrated at the assay temperature (EcLpxC at 30 °C, AaLpxC at 60 °C), and the reactions were initiated by the addition of enzyme. After incubation for various times, the reaction was quenched by the addition of sodium hydroxide, which also cleaves the myristate substituent for ease of separation. The substrate and product were separated on PEI-cellulose TLC plates (0.1 m guanidinium HCl) and quantified by scintillation counting. The initial rate of product formation (<20% reaction) was determined from these data. For the EcLpxC mutants the initial rate was measured in 20 mm bis-Tris propane, pH 7.5, at seven to nine different concentrations (25 nm to 8 μm) of myr-UDP-GlcNAc. The steady-state parameters kcat, Km, and kcat/Km were obtained by fitting the Michaelis-Menten equation to the initial linear velocities measured at various substrate concentrations. For the pH dependence experiments, LpxC activity was assayed under kcat/Km conditions at several concentrations of myr-UDP-GlcNAc (50–200 nm) to demonstrate a linear dependence on substrate concentration ([S] < Km) in the pH range of 5.5–10.5. Wild-type and the mutant LpxC enzymes were linearly dependent on substrate concentration with the exception of E78A and E78A/H265A at high pH where little turnover was observed at concentrations below 200 nm, consistent with a Km > 200 nm. The buffers were either a combination of 100 mm acetate, 50 mm bis-Tris, 50 mm triethanolamine over the entire pH range or 20 mm MES, pH 5.5–6.5, 20 mm bis-Tris propane, pH 7–9.1, or 20 mm CAPS, pH 9.8–10.7. Equation 1 (two ionizations) was fit to the pH rate profiles. (kcatKm)obx=k1(1+[H]Ka1+Ka2[H]) (Eq. 1) Equation 2 was fit to the pH rate profiles for the E78A/H265A mutants, (kcatKm)obx=k1+k2([H]/Ka1)(1+[H]Ka1+Ka2[H]) (Eq. 2) where k1 is the kcat/Km at the pH optimum, and k2 is the pH independent value of kcat/Km at low pH. LpxC is stable over this pH range under the assay conditions. The catalytic activity of LpxC decreases ≤2-fold after incubation at the altered pH for ≤5 min followed by measuring the activity at pH 7.5. For the solvent isotope effect experiments, the initial rates at subsaturating substrate concentrations in H2O were compared with the initial rates in ∼95% D2O. The pD values obtained for the D2O buffers using the pH meter readings were corrected by adding 0.4 to these values (24Srere P.A. Kosicki G.W. Lumry R. Biochim. Biophys. Acta. 1961; 50: 184-&Crossref Scopus (22) Google Scholar). Crystallography—The C193A/ΔAsp284–Leu294 variant of A. aeolicus LpxC was used for crystallography experiments as described (15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar). Crystallization was achieved by equilibrating a hanging drop containing 3 μl of protein solution (3 mg/ml LpxC, 100 mm HEPES, pH 7.5, 180 mm NaCl, 9–14% PEG3350, and 0.5 mm ZnSO4) and 3 μl of precipitant buffer (100 mm HEPES, pH 7.5, 180 mm NaCl, 9–14% PEG3350, and 0.5 mm ZnSO4) over a reservoir containing ∼1 ml of precipitant buffer. Crystals with maximum dimensions of 0.3 × 0.15 × 0.15 mm3 grew within 3 days and were gradually transferred to a stabilization buffer of 100 mm sodium cacodylate, pH 6.0, 180 mm NaCl, 11–16% PEG 3350, 0.5 mm ZnSO4, and 1% glycerol. Crystals were flash-cooled in liquid nitrogen following cryoprotection with 22% glycerol and diffracted x-rays to 2.1 Å at the Argonne National Laboratory (IMCA-CAT, Argonne, IL). Crystals were isomorphous with those prepared at pH 7.0 (15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar) and belong to space group P61 with unit cell dimensions a = b = 101.3 Å, c = 122.7 Å. Data were indexed and merged using the program HKL2000 (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 306-326Google Scholar). The structure was solved by molecular replacement using the zinc-inhibited enzyme, excluding all zinc ions, solvent, and fatty acid molecules, as a search probe for rotation and translation functions calculated with the program AmoRe (26Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar). It was clear in initial electron density maps that the inhibitory metal ion, ZnB, had dissociated and that a tetrahedral cacodylate anion was coordinated to the catalytic metal ion ZnA. In native AaLpxC, a fatty acid interpretable as myristate or disordered palmitate occupied the hydrophobic tunnel and coordinated to ZnB; because fatty acids were not included in the crystallization medium, this fatty acid must be a remnant of heterologous expression in E. coli. The fatty acid remained bound in the LpxC-cacodylate complex, and its carboxylate group was displaced to two alternate conformations as a consequence of ZnB dissociation. Iterative cycles of refinement and model rebuilding were performed with the programs CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 55: 905-921Crossref Scopus (16919) Google Scholar) and O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar), respectively, to improve the structure as monitored by Rfree. Data collection and refinement statistics are reported in Table I.Table IData collection and refinement statisticsComplexLpxC-Cacodylate complexLpxC-Palmitate complexResolution range (Å)30-2.150-2.7Reflections (measured/unique)257,853/41,614114,604/19,665Completeness (%) (overall/outer shell)100/10098.2/100RmergeaRmerge = Σ|Ij - 〈Ij〉|ΣIj, where Ij is the observed intensity for reflection j and 〈Ij〉 is the average intensity calculated for reflection j from replicate data. (overall/outer shell)0.054/0.3590.115/0.335〈I/sigma〉 (overall/outer shell)18.1/3.110.4/4.7Protein atoms (no.) bPer asymmetric unit.42984298Solvent atoms (no.)bPer asymmetric unit.318117Glycerol molecules (no.)bPer asymmetric unit.1NAdNA, not applicable.Chloride ions (no.)bPer asymmetric unit.33Sulfate ions (no.)bPer asymmetric unit.2NAdNA, not applicable.Metal ions (no.)bPer asymmetric unit.55Ligand atoms (no.)bPer asymmetric unit.4636Reflections used in refinement (work/free)39,481/208817,605/1919R/RfreecR = Σ||Fo| - |Fc||/Σ|Fo|, where R and Rfree are calculated by using the working and test reflection sets, respectively.0.182/0.2130.201/0.239Root mean square deviationsBonds (Å)0.0050.006Angles (degree)1.21.2Proper dihedral23.523.3Improper dihedral0.70.8a Rmerge = Σ|Ij - 〈Ij〉|ΣIj, where Ij is the observed intensity for reflection j and 〈Ij〉 is the average intensity calculated for reflection j from replicate data.b Per asymmetric unit.c R = Σ||Fo| - |Fc||/Σ|Fo|, where R and Rfree are calculated by using the working and test reflection sets, respectively.d NA, not applicable. Open table in a new tab In a second experiment, crystals were gradually transferred to a stabilization buffer containing 100 mm bis-Tris, pH 6.0, 180 mm NaCl, 11–16% PEG 3350, 0.5 mm ZnSO4, and 1% glycerol. Following transfer to a 22% glycerol cryoprotectant, crystals were flash-cooled in liquid nitrogen and yielded x-ray diffraction data to 2.7 Å using an R axis IV++ image plate detector mounted on a Rigaku-200HB rotating anode x-ray generator. Diffraction data were indexed and merged using the program d*Trek (29Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). The structure of zinc-inhibited LpxC, excluding all zinc ions, solvent, and fatty acid molecules, was used as a search probe in molecular replacement calculations with the program EPMR (30Kissinger C.R. Gehlhaar D.K. Smith B.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (688) Google Scholar) to phase the initial electron density map. The atomic model was refined and rebuilt using the programs CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 55: 905-921Crossref Scopus (16919) Google Scholar) and O (28Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar), respectively. Strict NCS constraints were used during the initial stages of refinement and then relaxed into appropriately weighted restraints as indicated by Rfree as refinement progressed. Data collection and refinement statistics are reported in Table I. Figs. 2a, 6a, and 7a were prepared using the program Bobscript (31Esnouf R.M. J. Mol. Graph. Model. 1997; 15: 132-134Crossref PubMed Scopus (1794) Google Scholar).Fig. 7a, omit electron density map of the LpxC-palmitate complex (contoured at 4σ). Atoms are color-coded as follows: C = yellow, O = red, N = blue; zinc appears as a gray sphere. b, hydrogen bond and metal coordination interactions in the LpxC-palmitate complex. Distances are indicated in Ångstroms.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mutations Decrease Catalytic Activity—Previous mutagenesis experiments (17Jackman J.E. Raetz C.R.H. Fierke C.A. Biochemistry. 2001; 40: 514-523Crossref PubMed Scopus (45) Google Scholar) and the crystal structure of the zinc-inhibited LpxC (15Whittington D.A. Rusche K.M. Shin H. Fierke C.A. Christianson D.W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8146-8150Crossref PubMed Scopus (131) Google Scholar) have suggested that residues Glu78, His265, and Asp246 are important for catalytic activity. Therefore, the steady-state parameters Km, kcat, and kcat/Km were determined for the E78A, H265A, D246A, and E78A/H265A EcLpxC mutants and compared with the values obtained for WT EcLpxC (Table II). The E78A, D246A, and H265A single mutations all decreased kcat/Km values by 400–2200-fold, whereas the largest decrease in kcat/Km was measured for the E78A/H265A double mutant (∼1.5 × 104-fold). The decrease in kcat/Km values observed for the LpxC mutants is predominantly explained by a decrease in the kcat values for these enzymes. The smallest decrease in kcat was observed for the E78A mutant (18-fold), whereas the largest decrease was observed for the E78A/H265A mutant (1.6 × 103-fold). These mutations have more modest effects on the Km values, with the E78A mutation causing the largest increase (23-fold). The observed changes in the Km values rule out the possibility that the activity in the mutants is because of WT contamination. To elucidate further the functional roles of these residues, we measured the pH dependence of catalysis.Table IISteady-state kinetic parameters for E. coli LpxC mutantsE. coli LpxCaThe metal-substituted enzymes were prepared with a stoichiometry of 1:1 as described under “Experimental Procedures.”,bThe initial rate for LpxC-catalyzed deacetylase activity was determined at 30 °C (20 mm bis-Tris propane, pH 7.5, 1 mg/ml bovine serum albumin, 0.5 mm triscarboxyethylphosphine) with myr-UDP-GlcNAc as the substrate. The kinetic parameters were obtained from the initial velocities, as described under “Experimental Procedures.”kcatKmkcat/KmFold decrease (kcat/Km)min-1μmμm-1 min-1WT-Zn2+90 ± 20.19 ± 0.01460 ± 10WT-Co2+27 ± 20.47 ± 0.0865 ± 0.67E78A5.0 ± 0.44.3 ± 0.61.16 ± 0.09400H265A0.30 ± 0.051.4 ± 0.50.21 ± 0.042190D246A0.094 ± 0.0090.25 ± 0.060.38 ± 0.061210E78A/H265A0.058 ± 0.0031.9 ± 0.20.031 ± 0.00214,800a The metal-substituted enzymes were prepared with a stoichiometry of 1:1 as described under “Experimental Procedures.”b The initial rate for LpxC-catalyzed deacetylase activity was determined at 30 °C (20 mm bis-Tris propane, pH 7.5, 1 mg/ml bovine serum albumin, 0.5 mm triscarboxyethylphosphine) with myr-UDP-GlcNAc as the substrate. The kinetic parameters were obtained from the initial velocities, as described under “Experimental Procedures.” Open table in a new tab pH Dependence of the LpxC-catalyzed Reaction—The pH dependence of the LpxC-catalyzed reaction for the EcLpxC and AaLpxC was determined under subsaturating substrate (kcat/Km) conditions to identify ionizations in the free enzyme or substrate that are important for catalytic activity. The kcat/Km values included the rate constants for substrate binding through formation of acetate (first irreversible step) (32Cleland W.W. Methods Enzymol. 1982; 87: 390-405Crossref PubMed Scopus (147) Google Scholar). In contrast, the kinetic parameter kcat included the rate constants after formation of the E·S complex through product dissociation. For enzymes wher" @default.
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