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• W2040215775 abstract "UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-l-alanyl-d-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-l-Ala-γ-d-Glu-meso-A2pm, has been solved to 2.0 Å resolution. Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium. The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-l-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of d-glutamic acid in the biosynthesis of the peptidoglycan precursor. The refined model reveals the binding site for UDP-MurNAc-l-Ala-γ-d-Glu-meso-A2pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism. Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site. In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified. UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-l-alanyl-d-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-l-Ala-γ-d-Glu-meso-A2pm, has been solved to 2.0 Å resolution. Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium. The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-l-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of d-glutamic acid in the biosynthesis of the peptidoglycan precursor. The refined model reveals the binding site for UDP-MurNAc-l-Ala-γ-d-Glu-meso-A2pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism. Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site. In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified. UDP-N-acetylmuramoyl-l-alanine: D-glutamate ligase folylpoly-γ-l-glutamate synthetase diaminopimelic acid selenomethionyl-MurE UDP-N-acetylmuramoyl-l-Ala-d-Glu UDP-N-acetylmuramoyl-l-Ala-γ-d-Glu-meso-A2pm root mean square Peptidoglycan, the polymeric mesh of the bacterial cell wall, plays a critical role in protecting bacteria against osmotic lysis. It consists of linear repeating disaccharide chains cross-linked by short peptide bridges. During the cytoplasmic steps involved in the biosynthesis of the peptidoglycan precursor, four ADP-forming ligases (namely the Mur ligases) catalyze the assembly of the peptide moiety by the successive addition of l-alanine,d-glutamate, diaminopimelic acid, or l-lysine, and, finally, dipeptide d-alanyl-d-alanine to UDP-N-acetylmuramic acid (1Bugg T.D. Walsh C.T. Nat. Prod. Rep. 1992; 9: 199-215Crossref PubMed Scopus (298) Google Scholar, 2van Heijenoort J. Cell Mol. Life Sci. 1998; 54: 300-304Crossref PubMed Scopus (68) Google Scholar). Because all these enzymes are essential for cell viability, they are attractive targets for antibacterial chemotherapy. In Escherichia coli, these ligases are the products of the murC, murD,murE, and murF genes, located in themra region (3Mengin-Lecreulx D. Ayala J. Bouhss A. van Heijenoort J. Parquet C. Hara H. J. Bacteriol. 1998; 180: 4406-4412Crossref PubMed Google Scholar). Sequence comparison of the four E. coli Mur ligases shows several homologous regions, suggesting that these enzymes may be evolutionarily related and may use similar enzymatic mechanisms (4Ikeda M. Wachi M. Jung H.K. Ishino F. Matsuhashi M. J. Gen. Appl. Microbiol. 1990; 36: 179-187Crossref Scopus (34) Google Scholar, 5Bouhss A. Mengin-Lecreulx D. Blanot D. van Heijenoort J. Parquet C. Biochemistry. 1997; 36: 11556-11563Crossref PubMed Scopus (86) Google Scholar, 6Eveland S.S. Pompliano D.L. Anderson M.S. Biochemistry. 1997; 36: 6223-6229Crossref PubMed Scopus (104) Google Scholar). In earlier publications, we reported the structure of UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD),1 both in the native form and complexed with substrates (7Bertrand J.A. Auger G. Fanchon E. Martin L. Blanot D. van Heijenoort J. Dideberg O. EMBO J. 1997; 16: 3416-3425Crossref PubMed Scopus (130) Google Scholar, 8Bertrand J.A. Auger G. Martin L. Fanchon E. Blanot D. Le Beller D. van Heijenoort J. Dideberg O. J. Mol. Biol. 1999; 289: 579-590Crossref PubMed Scopus (131) Google Scholar, 9Bertrand J.A. Fanchon E. Martin L. Chantalat L. Auger G. Blanot D. van Heijenoort J. Dideberg O. J. Mol. Biol. 2000; 301: 1257-1266Crossref PubMed Scopus (77) Google Scholar). MurD consists of three domains with topologies reminiscent of a nucleotide-binding fold; the N- and C-terminal domains have a dinucleotide-binding fold (the Rossmann fold), and the central domain displays a mononucleotide-binding fold, also seen in ATP-binding proteins. A comparison of six MurD structures reveals that large C-terminal rotation, loop rearrangement, and subdomain movements occur upon substrate binding (9Bertrand J.A. Fanchon E. Martin L. Chantalat L. Auger G. Blanot D. van Heijenoort J. Dideberg O. J. Mol. Biol. 2000; 301: 1257-1266Crossref PubMed Scopus (77) Google Scholar). In addition, several potentially important residues for substrate binding and/or catalysis have been identified (10Bouhss A. Dementin S. Parquet C. Mengin-Lecreulx D. Bertrand J.A. Le Beller D. Dideberg O. van Heijenoort J. Blanot D. Biochemistry. 1999; 38: 12240-12247Crossref PubMed Scopus (51) Google Scholar). Recently, the x-ray structure of the folylpoly-γ-l-glutamate synthetase (FGS) of Lactobacillus caseihas been reported (11Sun X. Bognar A.L. Baker E.N. Smith C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6647-6652Crossref PubMed Scopus (70) Google Scholar). Despite low sequence identity, FGS and MurD are clearly both members of the Mur ADP-forming ligase superfamily (9Bertrand J.A. Fanchon E. Martin L. Chantalat L. Auger G. Blanot D. van Heijenoort J. Dideberg O. J. Mol. Biol. 2000; 301: 1257-1266Crossref PubMed Scopus (77) Google Scholar,12Sheng Y. Sun X. Shen Y. Bognar A.L. Baker E.N. Smith C.A. J. Mol. Biol. 2000; 302: 425-438Crossref Scopus (32) Google Scholar). In E. coli, MurE catalyzes the addition ofmeso-diaminopimelic acid (meso-A2pm) to the nucleotide precursor, UDP-MurNAc-l-Ala-d-Glu (UMAG), according to the reaction: UMAG + meso-A2pm + ATP ↔ UDP-MurNAc-l-Ala-γ-d-Glu-meso-A2pm (UMT) + ADP + Pi. As established with other Mur ligases, the MurE reaction presumably proceeds by phosphorylation of the C-terminal carboxylate of UMAG by the γ-phosphate of ATP to form an acyl phosphate intermediate, followed by nucleophilic attack by the α-amino group of A2pm to produce UMT (Fig.1), ADP, and inorganic phosphate (13Falk P.J. Ervin K.M. Volk K.S. Ho H.T. Biochemistry. 1996; 35: 1417-1422Crossref PubMed Scopus (68) Google Scholar, 14Vaganay S. Tanner M.E. van Heijenoort J. Blanot D. Microbial Drug Resistance. 1996; 2: 51-54Crossref PubMed Scopus (38) Google Scholar, 15Bouhss A. Dementin S. van Heijenoort J. Parquet C. Blanot D. FEBS Lett. 1999; 453: 15-19Crossref PubMed Scopus (28) Google Scholar). This mechanism is supported by the three-dimensional structures of MurD and MurD complexes. Some bacteria (such as E. coli and Bacillus subtilis) containmeso-A2pm, and others (such asStreptococcus pneumoniae and Staphylococcus aureus) contain l-lysine at the third position of the peptide side chain of cell wall peptidoglycan. In each case, the MurE enzymes have been shown to efficiently discriminate between these two amino acids in vitro, because they are only able to catalyze the addition of either meso-A2pm orl-lysine to UMAG (16Ito E. Strominger J.L. J. Biol. Chem. 1973; 248: 3131-3136Abstract Full Text PDF PubMed Google Scholar, 17Mengin-Lecreulx D. Blanot D. van Heijenoort J. J. Bacteriol. 1994; 176: 4321-4327Crossref PubMed Google Scholar). Because these two amino acids effectively coexist in bacterial cells, the high specificity of the MurE enzymes acts as a gatekeeper to ensure that only the specific substrate is incorporated in the peptidoglycan presursor. However, this difference in specificity is not clearly reflected in the protein sequence, because only 28 and 32‥ identity is seen between theE. coli and S. pneumoniae or E. coliand B. subtilis sequences, respectively. This report describes the crystal structure of E. coli MurE in the presence of the UMT reaction product. The structure reveals that the enzyme has a three-domain topology and allows the localization of the active site and the identification of the residues involved in UMT binding. In addition, we compare and discuss the two structurally characterized ligases, MurD and MurE. DNA restriction enzymes and synthetic oligonucleotides were obtained from Eurogentec or New England Biolabs. Polymerase chain reaction amplification of DNA was performed in a Thermocycler 60 apparatus (Bio-med) using Taq polymerase (Appligene), and DNA fragments were purified using the Wizard purification system.meso-[14C]A2pm was obtained from the Commissariat à l'Energie Atomique (Saclay, France). UMAG was synthesized from UDP-N-acetylmuramoyl-l-Ala, using purified MurD (18Auger G. Martin L. Bertrand J. Ferrari P. Fanchon E. Vaganay S. Pétillot Y. van Heijenoort J. Blanot D. Dideberg O. Protein Expression Purif. 1998; 13: 23-29Crossref PubMed Scopus (46) Google Scholar), and UMT was prepared as previously described (19Flouret B. Mengin-Lecreulx D. van Heijenoort J. Anal. Biochem. 1981; 114: 59-63Crossref PubMed Scopus (64) Google Scholar). E. coli strains JM83 (ara Δ [lac-proAB] rpsL thi φ80 dlacZ Δ M15) (20Yanisch-Perron C. Vieira J. Messing J. Gene ( Amst. ). 1985; 33: 103-119Crossref PubMed Scopus (11472) Google Scholar) and B180 (ΔmetA::Cmr) (21Richaud C. Mengin-Lecreulx D. Pochet S. Johnson E.J. Cohen G.N. Marlière P. J. Biol. Chem. 1993; 268: 26827-26835Abstract Full Text PDF PubMed Google Scholar) were used as plasmid hosts and for the preparation of the overproduced His6-tagged MurE enzyme. The pTrcHis60 plasmid, a pTrc99A (Amersham Pharmacia Biotech) derivative for the production of C-terminal His6-tagged proteins, has been recently described (22Pompeo F. van Heijenoort J. Mengin-Lecreulx D. J. Bacteriol. 1998; 180: 4799-4803Crossref PubMed Google Scholar). 2YT medium (23Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) or M9 minimal medium (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was used for growing cells, with growth being monitored at 600 nm with a Shimadzu UV-1601 spectrophotometer. For strains carrying drug resistance genes, the antibiotics used were ampicillin (100 μg·ml−1) and chloramphenicol (25 μg·ml−1). Small and large scale plasmid isolations were carried out by the alkaline lysis method, and standard procedures were used for endonuclease digestions, ligation, and agarose electrophoresis (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). E. coli cells were made competent and transformed with plasmid DNA using the method of Dagert and Ehrlich (25Dagert M. Ehrlich S.D. Gene ( Amst. ). 1979; 6: 23-28Crossref PubMed Scopus (849) Google Scholar). A plasmid suitable for high level overproduction of MurE (C-terminal His6-tagged form) was constructed as follows. Polymerase chain reaction primers were designed to incorporate a NcoI site (shown in boldface type) 5′ to the initiation codon (underlined) of murE, 5′-GGGACCCATGGCAGATCGTAATTTGCGCGAC-3′, and a BglII site (in boldface type) 3′ to the gene without its stop codon, 5′-TACGCAGATCTTGCAATCACCCCCAGCAG-3′. These primers were used to amplify the murE gene from the E. coli chromosome, and then the resulting material was treated with NcoI and BglII and ligated between the same sites of vector pTrcHis60, resulting in pMLD117, a plasmid allowing expression of the gene under the control of the strong isopropyl-β-d-thiogalactopyranoside-inducibletrc promoter. JM83 (pMLD117) cells were grown exponentially at 37 °C in 2YT-ampicillin medium (18 liters of culture in a fermenter). At an optical density of 0.1, isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 1 mm, and growth was continued for 3 h (final optical density was 1.3). The cells were harvested at 4 °C (about 45 g of wet weight), and the cell pellets were washed with cold 20 mm potassium phosphate buffer, pH 7.4, containing 1 mm β-mercaptoethanol and 0.5 mmMgCl2 (buffer A) and then stored at −20 °C. Cells from one-fifth of the preparation were suspended in 9 ml of buffer A and disrupted by sonication at 4 °C using a Bioblock Vibracell 72412 sonicator. The resulting suspension was centrifuged at 4 °C for 20 min at 200,000 × g, and the pellet was discarded. SDS-polyacrylamide gel electrophoresis analysis showed that MurE accounted for about 20‥ of the crude protein soluble extract (data not shown). For the preparation of the MurE protein in which all methionine residues were replaced by selenomethionine (SeMurE), E. colimethionine-auxotrophic strain β180 was transformed with plasmid pMLD117, and the resulting strain was cultured in M9 medium (2 liter culture) supplemented with 0.4‥ glucose, ampicillin, and all the usual amino acids (100 μg·ml−1), except thatl-selenomethionine was substituted forl-methionine. Expression of murE was induced at mid-log phase (optical density was 0.6) with 1 mmisopropyl-β-d-thiogalactopyranoside, and cell growth was allowed to continue for a further 3 h (final optical density was 1.8). The cells were then harvested, washed in buffer A, and disrupted by sonication, and the crude protein extract was prepared as described above. The His6-tagged proteins (MurE and SeMurE) were purified on Ni2+-nitrilotriacetate-agarose essentially following the manufacturer's recommendations (Qiagen): binding to the resin, washing with buffer A containing 20 mm imidazole and 300 mm NaCl, and elution of the adsorbed proteins by a discontinuous gradient of 20–300 mm imidazole (MurE and SeMurE behaved similarly and eluted at a concentration of imidazole of about 150 mm). The pooled fractions were dialyzed against 20 mm HEPES buffer, pH 7.4, containing 5 mmdithiothreitol and 200 mm NaCl and then concentrated on PM10 membranes (Millipore) to ∼12 mg·ml−1 for use in crystallization experiments. SDS-polyacrylamide gel electrophoresis analysis of proteins performed on 12‥ polyacrylamide gels (26Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3025) Google Scholar) showed that the His6-tagged proteins were at least 90‥ pure. Protein concentrations were determined by the Bradford method (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar), using bovine serum albumin as a standard. Typically, 15–20 mg of pure protein (MurE or SeMurE) were obtained per liter of culture. Amino acid and mass spectrometry (matrix-assisted laser desorption/ionization) analyses confirmed the composition and molecular mass of the two proteins as well as the 100‥ selenomethionine substitution in SeMurE. The meso-A2pm adding activity was assayed by measuring the formation of radioactive UMT. The reaction mixture (0.1 m Tris-HCl buffer, pH 8.6, 0.1m MgCl2, 5 mm ATP, 0.2 mm UMAG, and 0.1 mm meso-[14C]A2pm (1.5 KBq); total volume, 75 μl) was incubated for 30 min at 37 °C. The reaction was stopped by addition of 10 μl of glacial acetic acid and the radioactive substrate and product were separated by reverse-phase high pressure liquid chromatography on a Nucleosil 5C18column (4.6 × 150 mm; Alltech France, Templemars, France) using 50 mm ammonium formate buffer, pH 3.9, at a flow rate of 0.6 ml·min−1. Detection was performed with a radioactive flow detector (model LB506-C1; EG&G Wallac/Berthold, Evry, France) using the Quicksafe Flow 2 scintillator (Zinsser Analytic, Maidenhead, UK), and quantification was carried out using the Winflow software (EG&G Wallac/Berthold). The activity of the pure preparation of His6-tagged MurE enzyme was 625 nmol ofmeso-A2pm incorporated in UMT per min and mg of protein. MurE, expressed as a C-terminal His6-tagged protein, consists of 502 amino acids (molecular mass was 54,278.5 Da). Crystals of native and selenomethionyl MurE were grown in the presence of the reaction product, UMT. Drops of 2 μl of protein solution (10 mg·ml−1 of purified enzyme, 20 mm HEPES, pH 7.5, 200 mm NaCl, 5 mm dithiothreitol, and 1 mm UMT) and 2 μl of reservoir buffer (0.1 mHEPES, pH 7.5, 13‥ polyethylene glycol monomethyl ether 5,000, 0.5m LiCl, 10‥ isopropanol, and 5 mmdithiothreitol) were equilibrated against 1 ml of reservoir buffer. The crystals, space group C2221, had unit cell dimensions ofa = 93.27 Å, b = 99.51 Å, andc = 234.34 Å, with two molecules in the asymmetric unit. Multiwavelength anomalous dispersion data collection was carried out on a single flash cooled crystal at three wavelengths on a BM14 beam line (European Synchrotron Radiation Facility, Grenoble, France). The long c axis was aligned so that it was almost coincident with the spindle axis, and data were collected at 2.8 Å resolution using a MAR ccd detector. The range of data collection was determined using STRATEGY (28Ravelli R.B.G. Sweet R.M. Skinner J.M. Duisenberg A.J.M. Kroon J. J. Appl. Crystallogr. 1997; 30: 551-554Crossref Scopus (61) Google Scholar). Data were processed using DENZO (29Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The relevant statistics are given in TableI. Data at 2 Å resolution were collected using a MAR ccd on the ID14-EH2 beam line (European Synchrotron Radiation Facility, Grenoble, France), a 110° sweep being made in increments of 0.25°. Data were processed using XDS (30Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3243) Google Scholar).Table IData reduction and phasing statisticsInflection pointPeakRemoteHigh resolutionData collectionWavelength (Å)0.97910.97900.85500.933f′/f“−9.6/3.22−7.63/7.6−1.7/3.54Resolution (Å)2.82.82.82.0Total data280,818283,864287,064323,548Unique data27,25327,24627,10772,674Redundancy4.24.74.34.5Completeness (‥) 1-aNumbers in parentheses correspond to the last resolution shell.99.6 (98.7)99.7 (99.0)99.5 (96.5)98.3 (91.2)Rsym(‥) 1-aNumbers in parentheses correspond to the last resolution shell.3.94.13.86.6 (22)Ranom5.92.93.3PhasingFigure of merit before solvent flattening (2.8 Å)0.811-a Numbers in parentheses correspond to the last resolution shell. Open table in a new tab Multiwavelength anomalous dispersion data were input into the program SOLVE (31Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 571-579Crossref PubMed Scopus (91) Google Scholar). All 24 selenium positions in the asymmetric unit were found, and experimental phases were calculated from these using a multiple isomorphous approach. The noncrystallographic symmetry was defined using FINDNCS (32Collaborative Computational Project No. 4, Acta Crystallogr. Sect. D Biol. Crystallogr., 50, 1994, 760, 763.Google Scholar). Density modification (33Cowtan K. Main P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 487-493Crossref PubMed Scopus (309) Google Scholar) was then used to extend the experimental phases to 2 Å using noncrystallographic symmetry averaging, solvent flattening, and histogram matching. The resulting experimental map was of excellent quality. The majority of the model was traced automatically using wARP (34Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (484) Google Scholar). Refinement to 2 Å was carried out by sequential use of the Crystallography and NMR Systems program (35Adams P.D. Pannu N.S. Read R.J. Brünger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar), interspersed with computer graphics model building using O (36Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The final model consists of two molecules of MurE (992 visible residues, of which 10 have been modeled with double conformations), 141 ligand atoms, and 394 water molecules. The stereochemistry of the final model was evaluated using the PROCHECK program (37Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The coordinates of the MurE structure have been deposited with the Brookhaven Protein Data Bank (accession code 1e8c) and will be released 1 year after publication. The final model, which contains 992 residues, two UMT molecules, and 394 water molecules, had a crystallographic R factor of 20.2‥ (Rfree = 23.0‥; Ref. 38Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3872) Google Scholar) for all 72,674 reflections in the resolution range 46.7–2.0 Å (TableII). The root mean square (rms) deviations were 0.006 Å from ideal bond lengths and 1.3° from ideal bond angles. After density modification, the experimental density map was of good quality. Fig. 2 shows the chemical modification of Lys224 by covalent binding to three atoms (see “Discussion”). The asymmetric unit contains two molecules (A and B) of MurE, essentially identical in conformation. After superimposition, the rms deviation between 495 pairs of equivalent Cα was 0.45 Å. No symmetry was seen between the two molecules. The Ramachandran plot (39Ramachandran G.N. Ramakrishnan C. Sasisekharan V. J. Mol. Biol. 1963; 7: 95-99Crossref PubMed Scopus (2725) Google Scholar) for the present model showed all the nonglycine residues to be in allowed regions. The average temperature factors were slightly different for molecules A and B (29.9 and 31.4 Å2, respectively) but identical for the two UMT molecules (39.5 Å2). A few residues at either end of the polypeptide chain had no visible electron density and were therefore not included in the model. However, 25 and 20 residues (molecule A and B, respectively) clearly showed holes in the electron density map. The damage was produced by third generation synchrotron radiation (40Ravelli R.B. McSweeney S.M. Struct. Fold Des. 2000; 8: 315-328Abstract Full Text Full Text PDF Scopus (336) Google Scholar), the most frequent being decarboxylation of acidic residues. The side chains were built into the electron density, and a partial occupancy was assigned for the side chain atoms based on difference Fourier maps.Table IIStatistics of the final modelResolution range (Å)46.7–2.0(2.13–2.00)No. reflections72674(10782)Crystallographic R-value (‥)20.2(21.3)Free R-value (‥)23.0(24.9)Rms deviations from standard geometryBond lengths (Å)0.006Bond angles (°)1.3Dihedral angles (°)23.5Improper angles (°)0.78Number of nonhydrogen atomsProtein atoms7341UMT359Water molecules393Average Bvalues (Å2)Molecule AMolecule BProtein atoms29.931.4UMT39.539.5Water molecules38.5Rms deviations between molecules A and B (all Cα)0.43 for 495 atoms Open table in a new tab MurE consists of three globular domains formed from contiguous segments in the amino acid sequence (Fig. 3). Domain 1 comprises residues 1–88 and consists of a five-stranded β-sheet surrounded by two helices (Fig.4 a). Comparison of its structure with the data base of known protein structures carried out using the DALI server (41Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1291) Google Scholar) revealed no homology with known protein structures; the structure showing the greatest similarity is a fragment of the transferrin receptor (42Lawrence C.M. Ray S. Babyonyshev M. Galluser R. Borhani D.W. Harrison S.C. Science. 1999; 286: 779-782Crossref PubMed Scopus (259) Google Scholar) with a rms deviation of 3.9 Å for the 70 structurally equivalent Cα atoms. Domain 2 (Fig. 4 b) comprises residues 90–338 and consists of a central six-stranded parallel β-sheet surrounded by seven α-helices. The fold of the central β-sheet is similar to the classic “mononucleotide-binding fold” found in many ATP-binding proteins, and domain 2 will therefore be referred to as the ATP-binding domain. As expected, using DALI, the structures with the highest Z scores were the central domain of MurD (7Bertrand J.A. Auger G. Fanchon E. Martin L. Blanot D. van Heijenoort J. Dideberg O. EMBO J. 1997; 16: 3416-3425Crossref PubMed Scopus (130) Google Scholar) (Protein Data Bank code1uag; Z score = 20.0) and the N-terminal domain of FGS (11Sun X. Bognar A.L. Baker E.N. Smith C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6647-6652Crossref PubMed Scopus (70) Google Scholar) (Protein Data Bank code 1fgs; Z score = 9.7). Superposition of domain 2 on the corresponding domain of the two ligases gave an rms deviation of 3.1 Å for the 193 structurally equivalent Cαs for MurD and 3.5 Å for the 168 Cαs for FGS. The loop between β6 and α4 of the ATPase domain (also referred to as the P loop) is probably involved in ATP binding. Domain 3 (340) contains a six-stranded β-sheet with parallel strands (β17, β18, β19, β20, and β21) and an antiparallel strand (β16), and five surrounding α-helices (Fig. 4 c). Surprisingly, as already observed for the C-terminal domains of MurD and FGS, domain 3 contains a Rossmann fold (Z score = 10.9 and 10.8 for MurD and FGS, respectively). Moreover, superposition of domain 3 on the corresponding domain of the two ligases gave an rms deviation of 2.7 Å for the 119 structurally equivalents Cαs for MurD and 2.4 Å for the 107 Cαs for FGS. The large insertion (464) between strands β22 and β23 has no structural equivalent in MurD and FGS. The product, UMT, binds in the cleft between the three domains. The experimental electron density map showed almost all of both UMT molecules. In the early stage of refinement, only the A2pm density was difficult to interpret. As shown in Fig.5, the bound UMT makes many polar interactions with the protein. The domain 1 residues involved in UMT binding are located in the two loops connecting β1 with β2 and β2 with α2. The geometry of the uridine-ribose moiety of the two UMT molecules is C2′-endo for the ribose ring pucker (χ = −114.5°) and an anti-orientation about the glycosyl bond. The uracyl ring forms two hydrogen bonds with Ser28, these being between O2 and the main chain nitrogen and between N3 and Oγ. In addition, it is inserted between a salt bridge (between Asp27 and Arg29) and Tyr50. Interestingly, C5and C6 of the pyrimidine ring are exposed to the solvent, explaining how dihydrouridine can replace uridine in the nucleotide substrate with little effect on the Km (97 μm versus 55 μm for the natural substrate) (43Abo-Ghalia M. Michaud C. Blanot D. van Heijenoort J. Eur. J. Biochem. 1985; 153: 81-87Crossref PubMed Scopus (28) Google Scholar). The pyrophosphate of UMT interacts with the loop connecting β2 with α2 (residues 42–47). The protein-phosphate interactions are made mainly through hydrogen bonds with main chain nitrogens, the α-phosphate oxygens forming hydrogen bonds with Gln44 and Ala45, and the β-phosphate oxygens with His43. One of the β-phosphate oxygens forms hydrogen bonds with ND1 of His43; the latter is the only residue to balance the negative charges of the pyrophosphate. The N-acetylmuramic acid ring bridges the gap between domain 1 and the ATP-binding domain. Two groups of hydrogen bonds are important; Oδ1 of Asn156 interact with O4′, and Gln190 and Arg192 are in contact with the acetyl group. Interestingly, Arg192 forms three other hydrogen bonds with the carbonyl oxygen of l-Ala and Oε1 of d-Glu. In the ATP-binding domain, three loops are close to the product (β8-α5, β9-α6, and α6-β10). Finally, the A2pm-containing region of UMT forms five hydrogen bonds with the C-terminal domain, the atoms involved being located in two loops connecting secondary structure elements (β19-α14) and (β21-β22). These very important interactions are discussed below. An alignment analysis using the 20 MurE sequences currently available shows that 24 amino acid residues are strictly conserved; none of these are found in the N-terminal domain, whereas 14 occur in the ATP-binding domain and 10 are in the C-terminal domain. Fig. 6shows the sequence alignment; for clarity, only four representative MurE ligase sequences were used (E. coli, B. subtilis, S. aureus, and S. pneumoniae). Of the 24 strictly conserved residues, only three (Thr158, Ser185, and Arg192) interact with the product, UMT. Eight others (Gly115, Gly118, Lys119, Glu182, His210, Asn310, Arg341, and Asp356) are invariant residues conserved in all the members of" @default.
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• W2040215775 date "2001-04-01" @default.
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• W2040215775 title "Crystal Structure of UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-Diaminopimelate Ligase from Escherichia Coli" @default.
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• W2040215775 doi "https://doi.org/10.1074/jbc.m009835200" @default.
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