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• W2034281269 abstract "UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed β-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specific activities under standard assay conditions. The decrease in activity corresponds to decreased kcat/Km,UDP-GlcNAcvalues for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in kcat/Km,UDP-GlcNAcmainly reflects an increased Km,UDP-GlcNAc. His125 of E. coli LpxA may therefore function as a catalytic residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate. UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed β-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specific activities under standard assay conditions. The decrease in activity corresponds to decreased kcat/Km,UDP-GlcNAcvalues for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in kcat/Km,UDP-GlcNAcmainly reflects an increased Km,UDP-GlcNAc. His125 of E. coli LpxA may therefore function as a catalytic residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate. UDP-N-acetylglucosamine acyl carrier protein bovine serum albumin diethylpyrocarbonate dithiothreitol isopropyl-1-thio-β-d-galactopyranoside phosphate-buffered saline UDP-N-acetylglucosamine (UDP-GlcNAc)1 acyltransferase catalyzes the first step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide in Gram-negative bacteria (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1027) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (234) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2 nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar). This enzyme, the product of the lpxA gene (4Crowell D.N. Anderson M.S. Raetz C.R.H. J. Bacteriol. 1986; 168: 152-159Crossref PubMed Google Scholar, 5Coleman J. Raetz C.R.H. J. Bacteriol. 1988; 170: 1268-1274Crossref PubMed Google Scholar), transfers an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein (ACP) to the glucosamine 3-OH of UDP-GlcNAc (Fig. 1) (6Anderson M.S. Bulawa C.E. Raetz C.R.H. J. Biol. Chem. 1985; 260: 15536-15541Abstract Full Text PDF PubMed Google Scholar, 7Anderson M.S. Raetz C.R.H. J. Biol. Chem. 1987; 262: 5159-5169Abstract Full Text PDF PubMed Google Scholar, 8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar). The acylation of UDP-GlcNAc is characterized by an unfavorable equilibrium constant (∼0.01) (8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar). Therefore, the second reaction of lipid A biosynthesis, in which the LpxA product UDP-3-O-(R-3-hydroxyacyl)-GlcNAc is deacetylated (9Anderson M.S. Robertson A.D. Macher I. Raetz C.R.H. Biochemistry. 1988; 27: 1908-1917Crossref PubMed Scopus (61) Google Scholar, 10Young 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), is the first irreversible step of the pathway (Fig. 1). The deblocked amino group is then immediately acylated with another R-3-hydroxyacyl moiety (6Anderson M.S. Bulawa C.E. Raetz C.R.H. J. Biol. Chem. 1985; 260: 15536-15541Abstract Full Text PDF PubMed Google Scholar, 11Kelly 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). Mature lipid A is a disaccharide of 2,3-diacylated glucosamine units derived from UDP-2,3-diacylglucosamine (Fig. 1) (12Bulawa C.E. Raetz C.R.H. J. Biol. Chem. 1984; 259: 4846-4851Abstract Full Text PDF PubMed Google Scholar, 13Ray B.L. Painter G. Raetz C.R.H. J. Biol. Chem. 1984; 259: 4852-4859Abstract Full Text PDF PubMed Google Scholar). Escherichia coli lipid A is further phosphorylated at the 1 and 4′ positions and is acylated with laurate and myristate, respectively, at the R-3-hydroxyl groups of the 2′ and 3′R-3-hydroxyacyl chains (Fig. 1) (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2 nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 14Ray B.L. Raetz C.R.H. J. Biol. Chem. 1987; 262: 1122-1128Abstract Full Text PDF PubMed Google Scholar, 15Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). Lipid A is required for growth of E. coli and most other Gram-negative bacteria (16Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar, 17Onishi 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). Lipid A is also necessary for maintaining the integrity of the outer membrane as a barrier to toxic chemicals (18Vaara M. Antimicrob. Agents Chemother. 1993; 37: 2255-2260Crossref PubMed Scopus (135) Google Scholar, 19Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2 nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar). Furthermore, lipid A is a potent activator of innate immunity in animal systems (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2 nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 20Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1312) Google Scholar, 21Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1294) Google Scholar). The study of the enzymes involved in lipid A biosynthesis should therefore prove useful for the development of new anti-infective drugs (22Wyckoff 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). All enzymes involved in E. coli lipid A biosynthesis have now been identified, and their structural genes have been cloned (3Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2 nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 22Wyckoff 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, 23Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 24Babinski K.J. Raetz C.R.H. FASEB J. 1998; 12 (Abstr. A1288): L63Google Scholar). The only enzyme of the pathway for which an x-ray structure is available is LpxA (25Raetz C.R.H. Roderick S.L. Science. 1995; 270: 997-1000Crossref PubMed Scopus (292) Google Scholar). LpxA is a trimer of identical subunits, and it represents the first example of a protein with a left-handed, parallel β-helix as the predominating feature of its secondary structure (25Raetz C.R.H. Roderick S.L. Science. 1995; 270: 997-1000Crossref PubMed Scopus (292) Google Scholar). The 10 coils of the β-helix of E. coli LpxA are specified by 24 complete and six incomplete hexapeptide repeats, most of which are contiguous (25Raetz C.R.H. Roderick S.L. Science. 1995; 270: 997-1000Crossref PubMed Scopus (292) Google Scholar). Three repeats (18 amino acid residues) make up one turn of the β-helix. Many other bacterial and eucaryotic proteins contain similar contiguous hexapeptide repeats (26Vaara M. FEMS Microbiol. Lett. 1992; 97: 249-254Crossref Google Scholar). Three additional hexapeptide repeat proteins have recently been crystallized and shown to contain the same left-handed β-helix structure as seen in LpxA. These are a carbonic anhydrase from Methanosarcina thermophila (27Kisker C. Schindelin H. Alver B.E. Ferry J.G. Rees D.C. EMBO J. 1996; 15: 2323-2330Crossref PubMed Scopus (207) Google Scholar), a tetrahydrodipicolinate N-succinyltransferase from Mycobacterium bovis BCG (28Beaman T.W. Binder D.A. Blanchard J.S. Roderick S.L. Biochemistry. 1997; 36: 489-494Crossref PubMed Scopus (65) Google Scholar), and a xenobiotic acetyltransferase from Pseudomonas aeruginosa (29Beaman T.W. Sugantino M. Roderick S.L. Biochemistry. 1998; 37: 6689-6696Crossref PubMed Scopus (83) Google Scholar). Like LpxA, these enzymes are trimers. There are no structural clues to the location or mechanism of the LpxA active site, because the LpxA crystal structure was solved in the absence of substrates or inhibitors (25Raetz C.R.H. Roderick S.L. Science. 1995; 270: 997-1000Crossref PubMed Scopus (292) Google Scholar). However, the sequences of more than 15 LpxAs are now available. The amino acid residues conserved across all LpxAs cluster around a deep cleft located between adjacent subunits (Fig. 2), which contains multiple histidines and other basic residues. Because of its symmetry, LpxA has three such clefts (not all visible in Fig. 2). The ACP substrate of LpxA consists of 77 amino acid residues and is very acidic (30Vagelos P.R. Majerus P.W. Alberts A.W. Larrabee A.R. Ailhaud G.P. Fed. Proc. 1966; 25: 1485-1494PubMed Google Scholar, 31Kim Y. Prestegard J.H. Proteins. 1990; 8: 377-385Crossref PubMed Scopus (156) Google Scholar). Accordingly, it is plausible that the substrates R-3-hydroxymyristoyl-ACP and UDP-GlcNAc might bind to this basic cleft. Further evidence for the importance of the cleft comes from recent studies of the acyl chain length specificity of E. coli LpxA (32Wyckoff T.J.O. Lin S. Cotter R.J. Dotson G.D. Raetz C.R.H. J. Biol. Chem. 1998; 273: 32369-32372Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Substitution of glycine 173 (red residue in the shaded area of Fig. 2) with methionine switches the acyl chain length selectivity of E. coli LpxA from 14 carbons to 10 (32Wyckoff T.J.O. Lin S. Cotter R.J. Dotson G.D. Raetz C.R.H. J. Biol. Chem. 1998; 273: 32369-32372Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We now present chemical modification studies to demonstrate that histidine, lysine, and arginine residues are important in LpxA catalysis, whereas serine and cysteine residues are not. We also use site-directed mutagenesis to examine the effects of changing conserved histidine, lysine, and arginine residues. The combined findings support the view that His125 of E. coli LpxA is directly involved in catalysis, whereas other conserved basic residues may participate in substrate binding. We also show that high levels of homogeneous LpxA do not catalyze UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange. The results argue against a ping-pong mechanism with an acyl-enzyme intermediate but are consistent with the direct transfer of the acyl chain from R-3-hydroxyacyl-ACP to the glucosamine 3-OH of UDP-GlcNAc. [α-32P]UTP was purchased from NEN Life Science Products. Tryptone, yeast extract, brain heart infusion medium, and agar were from Difco. Antibiotics, glucosamine-1-phosphate, and ACP were products of Sigma. Chloroform, methanol, and acetic acid were from Mallinckrodt. All other chemicals were obtained from Sigma or Mallinckrodt. Silica gel-60 thin layer plates (0.25 mm) were purchased from Merck. Restriction enzymes, Klenow, and T4 DNA ligase were from New England BioLabs or Roche Molecular Biochemicals. Shrimp alkaline phosphatase was from U. S. Biochemical Corp. Primers for mutagenesis were custom-made by Life Technologies, Inc. The LpxC inhibitor L-573,655 (17Onishi 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) was provided by Dr. A. Patchett (Merck Research Laboratories, Rahway, NJ). The assay monitors the conversion of [α-32P]UDP-GlcNAc to [α-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc (7Anderson M.S. Raetz C.R.H. J. Biol. Chem. 1987; 262: 5159-5169Abstract Full Text PDF PubMed Google Scholar, 8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar). The standard reaction mixture (10–20 μl) contains 40 mm HEPES, pH 8, 1 mg/ml BSA, 10 μm R-3-hydroxymyristoyl-ACP, and 10 μm[α-32P]UDP-GlcNAc (2 × 106 cpm/nmol). Substrates were synthesized as described previously (33Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The reaction is started by the addition of enzyme (either purified or in a cell extract). The reaction mixture is incubated at 30 °C for 1–10 min. For measuring initial rates, the enzyme concentrations (monomer) are typically 1–10 nm. The reactions are terminated by spotting 2–2.5-μl portions onto a silica thin layer chromatography plate. After the spots air dry, the plates are developed in the solvent chloroform/methanol/water/acetic acid (25:15:4:2, v/v/v/v). The plates are dried and then exposed to imaging screens overnight at room temperature. The plates are visualized, and the extent of the reaction is quantified using a Molecular Dynamics PhosphorImager, operated with ImageQuant software. For assays of E. coli crude extracts, 0.2 mg/ml L-573,655 (17Onishi 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) from a 10 mg/ml stock in dimethyl sulfoxide is added to the assay mixtures to inhibit further metabolism of the LpxA reaction product by LpxC present in these extracts (Fig. 1). This is unnecessary for assays of purified LpxA or of Corynebacterium glutamicum extracts expressing recombinant E. coli LpxA. The assay of LpxA in the reverse direction monitors the conversion of [α-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc to [α-32P]UDP-GlcNAc (8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar, 34Williamson J.M. Anderson M.S. Raetz C.R.H. J. Bacteriol. 1991; 173: 3591-3596Crossref PubMed Google Scholar). The assay mixture (10–20 μl) includes 40 mm HEPES, pH 8, 1 mg/ml BSA, 10 μm acyl acceptor, 10 μm LpxA (monomer), and 80 nm[α-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc (7 × 108 cpm/nmol) (35Sorensen 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) (provided by Jane E. Jackman, Duke University). ACP, coenzyme A (Sigma), pantetheine (Sigma), N-acetylcysteamine (Aldrich), glutathione (Sigma), and UDP-GlcNAc were tested as acyl acceptors in the reverse direction. All the acyl acceptors are reduced with a 10-fold excess of dithiothreitol (DTT) for 20 min at room temperature prior to the assay. The reaction is started by addition of purified LpxA and incubated at 30 °C. Reactions are terminated by spotting 2 μl onto a silica thin layer chromatography plate. Plates are developed and quantified as described above. For assays of cell free extracts, 50-ml cultures of BL21(DE3)/pLysE strains carrying plasmids containing wild type and mutant lpxA genes were grown at 37 °C in LB (10 g/liter tryptone, 5 g/liter yeast extract, 10 g/liter NaCl) (36Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) with 100 μg/ml ampicillin (225 rpm). At A600 = 0.6, they were induced with 1 mm IPTG and grown for 3 more hours. Cells were washed once with 5 ml of 10 mm potassium phosphate, pH 7, containing 0.2 m NaCl and 20% glycerol, resuspended in 2 ml of the same, and stored at −80 °C. Cells were broken by one passage through a French pressure cell at 18,000 psi and centrifuged at 10,000 × g for 20 min to remove cell debris. Membrane-free supernatants were prepared by centrifugation at 150,000 × g for 90 min. For expression of LpxAs in the absence of chromosomally encoded residual activity, 500-ml cultures of C. glutamicum strains (37Brabetz W. Liebl W. Schleifer K.H. Arch. Microbiol. 1991; 155: 607-612Crossref PubMed Scopus (46) Google Scholar) carrying plasmids containing wild type and mutant lpxA genes were grown at 30 °C in LB broth supplemented with 5 μg/ml chloramphenicol and 50 μg/ml rifampicin to A600 = 1. Cells were washed once with 40 ml of 10 mm potassium phosphate, pH 7, containing 0.2m NaCl and 20% glycerol, resuspended in 2 ml of the same, and stored at −80 °C. Cells were broken by three passages through a French pressure cell at 18,000 psi and centrifuged at 10,000 ×g for 20 min to remove cell debris. Wild type E. coli LpxA was purified from BL21(DE3)/pLysE/pTO1 (33Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). pTO1 is a pET23c (Novagen) vector containing wild type E. coli lpxA. Mutant LpxAs were purified from BL21(DE3)/pLysE strains carrying pET23c based plasmids containing mutant lpxA genes (see below). BL21(DE3)/pLysE cultures (500 ml) were grown in LB with 100 μg/ml ampicillin at 37 °C (225 rpm), induced with 1 mm IPTG at A600 = 0.6, and grown for 3 more hours (final A600, ∼2). Cells were washed once with 50 ml of 10 mm potassium phosphate, pH 7, containing 0.2m NaCl and 20% glycerol, resuspended in 20 ml of the same, and stored at −80 °C. Cells were broken by one passage through a French pressure cell at 18,000 psi and centrifuged at 10,000 ×g for 20 min to remove cell debris. Membrane-free supernatants were prepared by centrifugation at 150,000 ×g for 90 min (final volume, 17 ml). A 40-ml Gel Matrex Green A (Amicon) column (8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar) was prepared by washing with 5 column volumes 8 m urea/0.5 m NaOH and equilibrating with 5 column volumes of 10 mm potassium phosphate, pH 7, containing 0.2 m NaCl and 20% glycerol. The membrane-free supernatant was diluted to 10 mg/ml (final volume, 25 ml) and applied to this column at 1.5 ml/min. The column was washed with the equilibration buffer until no more protein emerged as judged by A280. The protein was eluted with a 400-ml 0.2–2.5 m NaCl gradient (in 10 mm potassium phosphate, pH 7, containing 20% glycerol). LpxA elutes at approximately 0.6 m NaCl (8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar). Fractions containing LpxA were pooled and dialyzed against 2 × 2l portions of 20 mm Tris, pH 7.4, containing 20% glycerol. This pool was applied at 1 ml/min to an 8 ml Source 15Q (Amersham Pharmacia Biotech) column equilibrated in the same buffer. The column was washed in the equilibration buffer until no more protein eluted. The protein was then eluted with a 160 ml of 0–0.6 m NaCl gradient (in 20 mm Tris, pH 8, containing 20% glycerol). LpxA elutes at approximately 0.2 m NaCl. Fractions containing LpxA were pooled, divided into aliquots, and then stored at −80 °C. In the following experiments, LpxA concentrations are expressed as the amount of monomer/unit volume. For inhibition studies, 20 μmpurified LpxA was incubated at room temperature in 40 mmpotassium phosphate, pH 7, with various concentrations of DEPC (Sigma) for 10 min. DEPC solutions (in ethanol) were made each day just prior to an experiment. Exact DEPC concentrations were determined by reacting with an excess of imidazole (Sigma) and measuring the A240 (ε = 3200m−1) (38Miles E.W. Methods Enzymol. 1977; 47: 431-442Crossref PubMed Scopus (810) Google Scholar). DEPC reactions with LpxA at room temperature were quenched by a 1:50 dilution into 70 μmimidazole (on ice) (at least a 10-fold molar excess over the DEPC). DEPC, ethanol, and imidazole at the concentrations used have no effect on the substrates of the LpxA reaction, and ethanol and imidazole have no effect on LpxA activity at the levels at which they are carried over into the assay mixture (not shown). The LpxA/DEPC/imidazole mixtures were further diluted with 1 mg/ml BSA and assayed by the standard method using 40 mm potassium phosphate, pH 7, instead of 40 mm HEPES, pH 8, but this modification has little effect on LpxA activity. For substrate protection studies, 20 μm purified LpxA was incubated at room temperature for 10 min in 40 mm potassium phosphate, pH 7, and 200 μm DEPC in the presence of 20 mm UDP-GlcNAc, or 40 μm R-3-hydroxymyristoyl-ACP. These concentrations correspond to about 20 times the Km (8Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar) for each substrate. The reactions were quenched by a 1:50 dilution into 40 μmimidazole (a 10-fold molar excess). The mixtures were then further diluted as appropriate into 1 mg/ml BSA and assayed by the standard method with 40 mm potassium phosphate, pH 7, taking into account the residual substrate concentrations carried over from the preincubation. For NH2OH reactivation studies, 20 μmpurified LpxA was incubated with and without 100 μm DEPC for 5 min at room temperature in 40 mm potassium phosphate, pH 7. The reactions were quenched by a 1:50 dilution into 20 μm imidazole. These mixtures were then diluted 1:2 into 40 mm potassium phosphate, pH 7, and 1 mg/ml BSA or into the same containing a final concentration of 10 mmNH2OH (Sigma). A 100 mm stock of NH2OH in water was made fresh each day. At various times, portions were diluted further into 1 mg/ml BSA as appropriate and assayed by the standard method with 40 mm potassium phosphate, pH 7. For spectrophotometric quantification of the extent of chemical modification, 20 μm purified LpxA was incubated with 20–90 μm DEPC for 15 min at room temperature. During the reaction, the A240 was followed to determine the concentration of modified histidines, and the A280 was also measured to exclude major structural changes in protein folding. At various times throughout the reaction, 3-μl portions were withdrawn and quenched with a 10-fold excess of imidazole in 1 mg/ml BSA. These sample were kept on ice until the end of the study, at which time all of the samples were assayed for LpxA activity by the standard method with 40 mm potassium phosphate, pH 7. In all experiments, 20 μm purified LpxA (monomer) was incubated at room temperature for 30 min with various concentrations of pyridoxal 5′-phosphate, phenylglyoxal, or phenylmethanesulfonyl fluoride. Pyridoxal 5′-phosphate reactions were quenched by a 1:2 dilution into 10 mm sodium borohydride (corresponding to at least a 10-fold molar excess over pyridoxal 5′-phosphate). All reactions were then diluted appropriately and assayed for LpxA activity. Pyridoxal 5′-phosphate (Sigma) was prepared in 40 mm HEPES, pH 8, whereas phenylglyoxal and sodium borohydride were dissolved in water, and the phenylmethanesulfonyl fluoride stock solution was prepared in isopropanol. None of these reagents has any effect on the substrates of the LpxA reaction, and isopropanol and sodium borohydride have no effect on LpxA activity at the levels carried over into the assay system. The pyridoxal 5′-phosphate and phenylglyoxal studies were done in 40 mmHEPES, pH 8 (39Matsuyama T. Soda K. Fukui T. Tanizawa K. J. Biochem. (Tokyo). 1992; 112: 258-265Crossref PubMed Scopus (17) Google Scholar, 40Matthews K.S. Chakerian A.E. Gardner J.A. Methods Enzymol. 1991; 208: 468-496Crossref PubMed Scopus (15) Google Scholar). The phenylmethanesulfonyl fluoride studies were done in 40 mm potassium phosphate, pH 7 (41Jauhiainen M. Dolphin P.J. J. Biol. Chem. 1986; 261: 7032-7043Abstract Full Text PDF PubMed Google Scholar). In all experiments, 10 μm purified LpxA (monomer) was incubated at room temperature for 30 min with various concentrations of methyl methane thiosulfonate or N-ethyl maleimide. Both reagents were from Sigma, and stock solutions were prepared in water. After 30 min, samples were diluted with 1 mg/ml BSA and assayed by the standard method with 40 mm potassium phosphate, pH 7. Recombinant DNA techniques were carried out as described by Sambrook et al. (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmid DNA was prepared using the Qiagen Spin Miniprep kit or the Bigger Prep Plasmid DNA Preparation Kit (5 Prime → 3 Prime, Inc., Boulder, CO). Restriction endonucleases, Klenow, T4 DNA ligase, and shrimp alkaline phosphatase were used according to the manufacturers' specifications. DNA was extracted from gels using the GeneClean kit (Bio 101, Inc.) according to the manufacturer's directions. LpxA mutants were made by the method of Kunkel et al. (43Kunkel T.A. Bebenek K. MacClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (632) Google Scholar) using pTO1 (33Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) as template. Plasmid pTO1 contains wild type E. coli lpxA in a pET23c (Novagen) vector. Resulting plasmids are named for the mutated gene they carry, prefaced by pTO, e.g. pTO-H122A. Each mutated gene was sequenced to ensure that only the desired mutation was present. Plasmids were transformed into CaCl2 competent BL21(DE3)/pLysE (Novagen) cells. pET23c-based plasmids containing wild type and mutant lpxA genes were digested with Nde I and Bam HI to excise the lpxA gene. This fragment was treated with Klenow fragment and then ligated into a Bam HI-digested and Klenow fragment-treated pGK1 Corynebacterium/E. coli shuttle vector (37Brabetz W. Liebl W. Schleifer K.H. Arch. Microbiol. 1991; 155: 607-612Crossref PubMed Scopus (46) Google Scholar). The plasmid with wild type lpxA is pTO8 (32Wyckoff T.J.O. Lin S. Cotter R.J. Dotson G.D. Raetz C.R.H. J. Biol. Chem. 1998; 273: 32369-32372Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Other plasmids are named for the mutated gene that they carry prefaced by pGK, e.g. pGK-H122A. Plasmids were introduced into C. glutamicum R163 by electroporation (32Wyckoff T.J.O. Lin S. Cotter R.J. Dotson G.D. Raetz C.R.H. J. Biol. Chem. 1998; 273: 32369-32372Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 44Liebl W. Bayerl A. Schein B. Stillner U. Schleifer K.H. FEMS Microbiol. Lett. 1989; 65: 299-304Crossref Scopus (99) Google Scholar). Competent cells were made by growing a 500-ml culture of C. glutamicum in LB broth at 30 °C (225 rpm) to A600 = 0.2. Cells were washed twice with 15% glycerol in water and resuspended in 2.5 ml of the same. Electroporation was done under the following conditions: 50 μl of cells and 1 μl (1 μg) of DNA in a 0.2-cm cuvette (Bio-Rad) at 2.5 kV, 25 microfarad, and 200 Ω. Cells were allowed to recover for 2 h at 30 °C in 1 ml of brain heart infusion (BHI) medium and then p" @default.
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