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• W2017007825 abstract "MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM159 alanylated the peptidoglycan ϵ-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurMPn16 activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurMPn16 acylation activities were far lower than the alanylation activity of MurM159. The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM159 compared with MurMPn16 bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNAAla:tRNASer ratio in 159 and Pn16. Kinetic analysis revealed that MurM159 acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that ϵ-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TΨC loop stem of the tRNAAla. It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of β-lactams for treatment of pneumococcal infection. MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM159 alanylated the peptidoglycan ϵ-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurMPn16 activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurMPn16 acylation activities were far lower than the alanylation activity of MurM159. The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM159 compared with MurMPn16 bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNAAla:tRNASer ratio in 159 and Pn16. Kinetic analysis revealed that MurM159 acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that ϵ-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TΨC loop stem of the tRNAAla. It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of β-lactams for treatment of pneumococcal infection. The peptidoglycan in Streptococcus pneumoniae and other Gram-positive pathogens is composed of a carbohydrate polymer consisting of alternating residues of N-acetylglucosamine and N-acetylmuramic acid. Appended to the N-acetylmuramic acid residue is the “stem peptide” composed of up to five amino acids, l-alanyl-γ-d-glutamyl-l-lysyl-d-alanyl-d-alanine. The stem peptides are themselves cross-linked between the ϵ-amino group of the lysine of a pentapeptide and the carbonyl group of the fourth position d-alanine of an adjacent stem (1Bugg T.D.H. Pinto M. Comprehensive Natural Products Chemistry. 3. Elsevier, Oxford, UK1999: 241-294Google Scholar). The pneumococcal stem peptide is further modified in S. pneumoniae where the lysyl residue ϵ-amino group is substituted by a dipeptide branch consisting of l-alanine or l-serine followed invariably by l-alanine (2Garcia-Bustos J. Tomasz A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5415-5419Crossref PubMed Scopus (125) Google Scholar, 3Garcia-Bustos J.F. Chait B.T. Tomasz A. J. Bacteriol. 1988; 170: 2143-2147Crossref PubMed Google Scholar, 4Severin A. Tomasz A. J. Bacteriol. 1996; 178: 168-174Crossref PubMed Google Scholar). The stem peptide is constructed in the cytoplasm appended to a UDP nucleotide (Fig. 1) in a series of reactions catalyzed by MurA to F, where MurC, -D, -E, and -F are responsible for the ATP-dependent ligation of l-alanine, d-glutamate, l-lysine, and d-alanyl-d-alanine, respectively, to form UDP-N-acetylmuramyl-l-alanyl-γ-d-glutamyl-l-lysyl-d-alanyl-d-alanine (UDP-MurNAcAEKAA) (1Bugg T.D.H. Pinto M. Comprehensive Natural Products Chemistry. 3. Elsevier, Oxford, UK1999: 241-294Google Scholar). The phospho-N-acetylmuramyl pentapeptide is transferred from this species by MraY to a membrane-bound undecaprenyl-phosphate carrier to form lipid I (undecaprenyl-pyrophosphoryl-N-acetylmuramyl-l-alanyl-γ-d-glutamyl-l-lysyl-d-alanyl-d-alanine), which is then glycosylated with UDP-N-acetylglucosamine by MurG to form lipid II (undecaprenyl-pyrophosphoryl-N-acetylmuramyl (N-acetylglucosaminyl)-l-alanyl-γ-d-glutamyl-l-lysyl-d-alanyl-d-alanine) (1Bugg T.D.H. Pinto M. Comprehensive Natural Products Chemistry. 3. Elsevier, Oxford, UK1999: 241-294Google Scholar) (Fig. 1). The dipeptide branch is added to the stem peptide lysine at some point after the stem peptide is constructed (5Plapp R. Strominger J.L. J. Biol. Chem. 1970; 245: 3667-3674Abstract Full Text PDF PubMed Google Scholar, 6Matsuhashi M. Dietrich C.P. Strominger J.L. J. Biol. Chem. 1967; 242: 3191-3206Abstract Full Text PDF Google Scholar, 7Petit J.-F. Strominger J.L. Söll D. J. Biol. Chem. 1968; 243: 757-767Abstract Full Text PDF PubMed Google Scholar, 8Schneider T. Senn M.M. Berger-Bachii B. Tossi A. Sahl H.-G. Wiedmann I. Mol. Microbiol. 2004; 53: 675-685Crossref PubMed Scopus (140) Google Scholar, 9Hegde S. Schraeder T.E. J. Biol. Chem. 2001; 276: 6998-7003Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) (e.g. lipid II in Fig. 1). After transport to the outer face of the cytoplasmic membrane, lipid II is polymerized by transglycosylation. This nascent peptidoglycan is given structural rigidity by transpeptidation between the position 3 lysine (with or without a dipeptide branch) and the fourth position d-alanine of adjacent stem peptides (Fig. 1) (1Bugg T.D.H. Pinto M. Comprehensive Natural Products Chemistry. 3. Elsevier, Oxford, UK1999: 241-294Google Scholar). The pneumococcal genes encoding the enzymes that construct the dipeptide branch, MurM and MurN, add the first and second amino acids to the stem peptide lysine, respectively (10Filipe S.R. Tomasz A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4891-4896Crossref PubMed Scopus (147) Google Scholar, 11Filipe S.R. Pinho M.G. Tomasz A. J. Biol. Chem. 2000; 275: 27768-27774Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). S. pneumoniae has acquired related MurM sequences, within its genome by homologous recombination to create a family of mosaics of related murM genes (12Filipe S.R. Severina E. Tomasz A. J. Bacteriol. 2000; 182: 6798-6805Crossref PubMed Scopus (39) Google Scholar, 13Chesnel L. Carapito R. Croizé J. Dideberg O. Vernet T. Zapun A. Antimicrob. Agents Chemother. 2005; 49: 2895-2902Crossref PubMed Scopus (40) Google Scholar). This has endowed the resulting family of MurM variants with vastly differing levels of activity in vivo and differing amino acid specificities for incorporation of alanine and serine (2Garcia-Bustos J. Tomasz A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5415-5419Crossref PubMed Scopus (125) Google Scholar, 12Filipe S.R. Severina E. Tomasz A. J. Bacteriol. 2000; 182: 6798-6805Crossref PubMed Scopus (39) Google Scholar, 14Filipe S.R. Severina E. Tomasz A. J. Biol. Chem. 2001; 276: 39618-39628Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Although not absolutely essential for high level penicillin resistance in laboratory strains of S. pneumoniae, clinical strains of this organism depend upon the activity of MurM for high level penicillin resistance (14Filipe S.R. Severina E. Tomasz A. J. Biol. Chem. 2001; 276: 39618-39628Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 15Barcus V.A. Ghanekar K. Yeo M. Coffey T.J. Dowson C.G. FEMS Microbiol. Lett. 1995; 126: 299-303Crossref PubMed Google Scholar, 16Smith A.M. Klugman K.P. Antimicrob. Agents Chemother. 2001; 45: 2393-2396Crossref PubMed Scopus (70) Google Scholar). Despite the medical importance of this protein, knowledge of the enzyme biochemistry of MurM is sketchy and inferred. Unavailability of the peptidoglycan precursor substrates of MurM has previously restricted analysis of this protein to what can be deduced from molecular genetics, bioinformatics, and analysis of peptidoglycan precursor pools within S. pneumoniae. Recent successes in the in vitro synthesis of these precursors in our laboratory and elsewhere have, however, made the analysis of MurM enzymology a realistic proposition (17Reddy S.G. Waddell S.T. Kuo D.W. Wong K.K. Pompliano D.L. J. Am. Chem. Soc. 1999; 121: 1175-1178Crossref Scopus (41) Google Scholar, 18El Zoeiby A. Sanschagrin F. Havugimana P.C. Garnier A. Levesque R.C. FEMS Microbiol. Lett. 2001; 201: 229-235Crossref PubMed Google Scholar, 19Breukink E. van Heusden H.E. Vollmerhaus P.J. Swiezewska E. Brunner L. Walker S. Heck A.J.R. Kruijff de B. J. Biol. Chem. 2003; 278: 19898-19903Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). We have therefore carried out a characterization of the enzymatic properties of MurM from two clinical isolates of S. pneumoniae, one highly penicillin-resistant (159) and the other penicillin-sensitive (Pn16), that has allowed us in this paper to 1) confirm the type of enzymatic reaction carried out by MurM; 2) correlate the enzyme biochemistry of MurM with the final composition of the peptidoglycan of these two strains; 3) deduce the specificity of MurM for its peptidoglycan precursor substrates, allowing delineation of what is required for substrate binding by this enzyme; and 4) define those regions of the tRNAAla substrate of MurM that are required for binding and catalysis. RNA species corresponding to the sequence of the full-length pneumococcal tRNAAlaUGC isoacceptor GGG GCC UUA GCU CAG CUG GGA GAG CGC CUG CUU UGC ACG CAG GAG GUC AGC GGU UCG AUC CCG CUA GGC UCC ACC A and corresponding to the 3′-aminoacylation site, acceptor stem, and TΨC loop and stem of the full-length pneumococcal tRNAAlaUGC, GGG GCC UAG CGG UUC GAU CCC GCU AGG CUC CAC CA (RNA minihelix), were synthesized, purified, and supplied with a 5′-phosphorylation by Dharmacon Inc. S. pneumoniae Pn16 MurE (MurEPn16) and Pseudomonas aeruginosa MurA, MurB, MurC, MurD, MurE, and MurF were overexpressed and purified (18El Zoeiby A. Sanschagrin F. Havugimana P.C. Garnier A. Levesque R.C. FEMS Microbiol. Lett. 2001; 201: 229-235Crossref PubMed Google Scholar, 20Blewett A.M. Lloyd A.J. Echalier A. Fulop V. Dowson C.G. Bugg T.D.H. Roper D.I. Acta. Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 359-361Crossref PubMed Scopus (6) Google Scholar). Bacterial purine nucleoside phosphorylase (Sigma) was repurified (21Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Crossref PubMed Scopus (489) Google Scholar). Pig heart isocitrate dehydrogenase (NADP+; Sigma) was re-purified by elution from Sepharose 4B-Procion Blue MX2G with 1 mm NADPH. Undecaprenyl-MurNAc(GlcNAc)-l-alanyl-γ-d-glutamyl-l-lysyl(ϵN-l-alanine)-d-alanyl-d-alanine (lipid II-Ala) was a generous gift from Dr. G. dePascale (Warwick University). Other chemicals are recorded in the supplemental Materials and Methods and were sourced as in Ref. 22Lloyd A.J. Brandish P.E. Gilbey A.M. Bugg T.D.H. J. Bacteriol. 2003; 186: 1747-1757Crossref Scopus (72) Google Scholar or were from Sigma or Melford Laboratories Ltd. Details of E. coli strains and plasmids used in this study are indicated in supplemental Materials and Methods. Pn16 (110K/70) serotype 42, was isolated in Papua, New Guinea and was penicillin-sensitive (minimum inhibitory concentration <0.016 μg·ml-1) (23Whatmore A.M. Barcus V.A. Dowson C.G. J. Bacteriol. 1999; 181: 3144-3154Crossref PubMed Google Scholar). 159 serotype 19A was isolated in Hungary (15Barcus V.A. Ghanekar K. Yeo M. Coffey T.J. Dowson C.G. FEMS Microbiol. Lett. 1995; 126: 299-303Crossref PubMed Google Scholar) and was penicillin-resistant (minimum inhibitory concentration >16 μg·ml-1). Strains were propagated on brain heart infusion agar containing 5% (v/v) sheep blood at 37 °C in 5% (v/v) CO2 or in liquid medium in brain heart infusion broth at 37 °C in 5% (v/v) CO2. DNA was extracted from lawns of pneumococci on brain heart infusion blood agar as described (23Whatmore A.M. Barcus V.A. Dowson C.G. J. Bacteriol. 1999; 181: 3144-3154Crossref PubMed Google Scholar). Details of preparation of M. flavus membranes are recorded in supplemental Materials and Methods. Peptidoglycan was extracted from late exponential phase S. pneumoniae, purified, and digested with muramidase, and the resulting stem peptides were extracted and fractionated by reverse phase HPLC 2The abbreviations used are:HPLChigh pressure liquid chromatographyCHAPS3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acidMOPS4-morpholinepropanesulfonic acidDTTdithiothreitolDAPmeso-diaminopimelic acidAlaRSalanyl-tRNAAla synthetaseSerRSseryl-tRNASer synthetaseES-MSelectrospray-mass spectrometry. on a Vydac 218TP54 column (4Severin A. Tomasz A. J. Bacteriol. 1996; 178: 168-174Crossref PubMed Google Scholar, 24Garcia-Bustos J.F. Tomasz A. J. Bacteriol. 1987; 169: 447-453Crossref PubMed Google Scholar). Peptidoglycan fragment structural assignments were made according to Refs. 2Garcia-Bustos J. Tomasz A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5415-5419Crossref PubMed Scopus (125) Google Scholar, 3Garcia-Bustos J.F. Chait B.T. Tomasz A. J. Bacteriol. 1988; 170: 2143-2147Crossref PubMed Google Scholar, 4Severin A. Tomasz A. J. Bacteriol. 1996; 178: 168-174Crossref PubMed Google Scholar, 11Filipe S.R. Pinho M.G. Tomasz A. J. Biol. Chem. 2000; 275: 27768-27774Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar. high pressure liquid chromatography 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid 4-morpholinepropanesulfonic acid dithiothreitol meso-diaminopimelic acid alanyl-tRNAAla synthetase seryl-tRNASer synthetase electrospray-mass spectrometry. SDS-PAGE, N-terminal protein sequencing, protein assays, and Western blotting for histidine tags were performed according to Ref. 22Lloyd A.J. Brandish P.E. Gilbey A.M. Bugg T.D.H. J. Bacteriol. 2003; 186: 1747-1757Crossref Scopus (72) Google Scholar and references therein. To sequence the Pn16 and 159 murM genes, primers were designed using the S. pneumoniae R6 genome sequence (25Hoskins J. Alborn Jr., W.E. Arnold J. Blaszczak L.C. Burgett S. DeHoff B.S. Estrem S.T. Fritz L. Fu D.J. Fuller W. Geringer C. Gilmour R. Glass J.S. Khoja H. Kraft A.R. Lagace R.E. LeBlanc D.J. Lee L.N. Lefkowitz E.J. Lu J. Matsushima P. McAhren S.M. McHenney M. McLeaster K. Mundy C.W. Nicas T.I. Norris F.H. O'Gara M. Peery R.B. Robertson G.T. Rockey P. Sun P.M. Winkler M.E. Yang Y. Young-Bellido M. Zhao G. Zook C.A. Baltz R.H. Jaskunas S.R. Rosteck Jr., P.R. Skatrud P.L. Glass J.I. J. Bacteriol. 2001; 183: 5709-5717Crossref PubMed Scopus (609) Google Scholar) at the J. Craig Ventner Institute (formally The Institute for Genome Research) web site to amplify the region between 232 nucleotides 5′ to the initiator ATG (primer 1, supplemental Table 1) of the murM gene to 162 nucleotides 3′ of the murM TAA stop codon (primer 2, supplemental Table 1) by PCR. DNA sequence between these primers was amplified by Pwo DNA polymerase according to the manufacturer's instructions. and a product of the correct size (1.6 kb) was purified using a Qiagen spin column and sequenced in both directions. To construct an expression vector carrying a murM allele with a 3′ sequence encoding a hexahistidine (His6) peptide, 1.3-kb fragments containing the murM allele from Pn16 and 159 were amplified by PCR from the appropriate pneumococcal DNA. Because of sequence divergence at the 5′ end of the open reading frame between the murM alleles, a 5′ primer for each gene was designed incorporating an NdeI restriction site for amplification of murMPn16 and murM159, respectively (primers 3 and 4, supplemental Table 1). A single 3′ primer for the amplification of both alleles was designed to incorporate a 3′ XhoI site and eliminate the 3′ stop codon (primer 5, supplemental Table 1). On PCR with PWO polymerase, products of the correct mass for murMPn16 and murM159 were obtained, purified, restricted with NdeI and XhoI, and ligated into similarly restricted pET21b as described in Ref. 22Lloyd A.J. Brandish P.E. Gilbey A.M. Bugg T.D.H. J. Bacteriol. 2003; 186: 1747-1757Crossref Scopus (72) Google Scholar. Clones carrying the recombinant murMPn16 and murM159 genes were verified by sequencing, and one correct clone was retained for expression of each protein (pET21b::murMPn16 and pET21b::murM159). To overexpress the MurM proteins, 650-ml cultures of E. coli C41 (DE3)/pRIL, harboring either pET21b::murMPn16 or pET21b::murM159 in Luria Broth (LB) + 50 μg/ml carbenicillin + 30 μg/ml chloramphenicol, were grown at 37 °C to an A600 of 0.6-1.0, when MurM expression was induced by 0.5 mm isopropyl β-d-thiogalactopyranoside, concurrent with the growth temperature being reduced to 25 °C. E. coli cells were harvested after 4 h and washed at 4 °C in 50 mm HEPES, 1 mm MgCl2, pH 7.5, and 2 mm β-mercaptoethanol. Analysis of whole cells and subcellular fractions thereof by SDS-PAGE and Western blotting suggested that MurMPn16 and MurM159 were mostly insoluble but could be solubilized by 1 m NaCl. 3A. J. Lloyd, unpublished data. To purify MurMPn16 or MurM159, all steps were performed at <4 °C. Cell pellets suspended/g in 3 ml of 50 mm HEPES, 1 mm MgCl2, 0.5 mm EGTA, 2 mm β-mercaptoethanol, 0.2 mm phenylmethanesulfonyl fluoride, 1 μm leupeptin, 1 μm pepstatin, pH 7.5, + 2.5 mg ml-1 chicken egg white lysozyme (lysis buffer) were shaken for 30 min, disrupted by sonication, and centrifuged at 10,000 × g for 30 min. The 10,000 × g pellet was extracted in 50 mm sodium phosphate, 1 m NaCl, 0.5 mm EGTA, 2 mm β-mercaptoethanol, 1 μm leupeptin, 1 μm pepstatin, and 0.2 mm phenylmethanesulfonyl fluoride, pH 7.0, for 30 min and then centrifuged for 30 min at 100,000 × g. The supernatant was retained, and the 100,000 × g pellet was re-extracted as above, and the supernatants were combined. The supernatant was sequentially fractionated between 25 and 50% saturation ammonium sulfate and by gel exclusion chromatography on a 500-ml Sephacryl S-200 column in 50 mm NaH2PO4, 0.5 m NaCl, 0.2 mm phenylmethanesulfonyl fluoride, 1 μm leupeptin, 1 μm pepstatin, pH 7.0 (phosphate buffer). Fractions containing MurM by Western blot were further purified by immobilized metal affinity chromatography on a 25-ml column of cobalttalon resin (Clontech) in phosphate buffer. Once unbound proteins were eluted, MurM was eluted by a 0-0.2 m imidazole gradient. The purity and identity of the final products of these purifications were assessed by SDS-PAGE, Western blotting, and N-terminal sequencing. Using the pneumococcal sequences in Ref. 26Tettelin H. Nelson K.E. Paulsen I.T. Eisen J.A. Read T.D. Peterson S. Heidelberg J. DeBoy R.T. Haft D.H. Dodson R.J. Durkin S. Gwinn M. Kolonay J.F. Nelson W.C. Peterson J.D. Umayam L.A. White O. Salzberg S.L. Lewis M.R. Radune D. Holtzapple E. Khouri H. Wolf A.M. Utterback T.R. Hansen C.L. McDonald L.A. Feldblyum T.V. Angiuoli S. Dickinson T. Hickey E.K. Holt I.E. Loftus B.J. Yang F. Smith H.O. Venter J.C. Dougherty B.A. Morrison D.A. Hollingshead S.K. Fraser C.M. Science. 2001; 293: 498-506Crossref PubMed Scopus (1119) Google Scholar, the above genes were cloned and overexpressed, and their products were purified by immobilized metal affinity chromatography and anion exchange chromatography. Details of these procedures are given in supplemental Materials and Methods. Syntheses were conducted at 37 °C overnight in 2-ml volumes in air-tight tubes with no head space, in 50 mm HEPES, 1 mm dithiothreitol (DTT), 50 mm KCl, 10 mm MgCl2 adjusted to pH 7.5, 3.65 μm MurA, 7.24 μm MurB, 3.50 μm MurC, 9.91 μm MurD, 9.09 μm MurEPn16, 0.4 μmol·min-1·ml -1 NADP+-linked isocitrate dehydrogenase, 6.7 μmol·min-1·ml-1 rabbit muscle pyruvate kinase, 26.7 mm dl-isocitrate, 79.8 mm phosphoenolpyruvate, 13.3 mm UDP-GlcNAc, 0.1 mm NADPH, 5 mm ATP, 20 mm l-alanine, 22.9 mm d-glutamate and 15 mm l-lysine. Syntheses were conducted as for UDP-MurNAcAEK, except that the phosphoenolpyruvate concentration was 99.8 mm and the incubations also contained 15 mm d-alanyl-d-alanine and 9.09 μm MurF159. Syntheses were conducted as described for UDP-MurNAcAEKAA, except that lysine was replaced by 30 mm meso-diaminopimelic acid (DAP), and both MurEPn16 and MurF159 were replaced by P. aeruginosa MurE and MurF. In all cases, the UDP-MurNAc peptide product was freed from protein by centrifugation through a Mr 10,000 cutoff membrane, and the filtrate was fractionated on a 50-ml column of Source 30 Q anion exchange resin from which it was eluted using a 0-1 m ammonium acetate gradient at pH 7.5. Fractions containing the UDP-MurNAcAEK product were identified enzymatically utilizing MurF159 and by negative ion electrospray-mass spectrometry (ES-MS) for this and all other UDP-MurNAc peptides. All products were lyophilized three times versus water and stored in solution at -20 °C. All syntheses were conducted using Micrococcus flavus membranes essentially as described in Ref. 19Breukink E. van Heusden H.E. Vollmerhaus P.J. Swiezewska E. Brunner L. Walker S. Heck A.J.R. Kruijff de B. J. Biol. Chem. 2003; 278: 19898-19903Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar. The lipid I or lipid II products were purified as described previously (19Breukink E. van Heusden H.E. Vollmerhaus P.J. Swiezewska E. Brunner L. Walker S. Heck A.J.R. Kruijff de B. J. Biol. Chem. 2003; 278: 19898-19903Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). To assay lipid-linked precursors, 50 μl of lipid I or II species suspended in 50 mm HEPES, 10 mm MgCl2, 30 mm KCl, and 1.5% (w/v) CHAPS, pH 7.6, were added to 50 μl of 1 m HCl. Samples were boiled for 30 min and neutralized with 2 m NaOH. The phosphate released was assayed according to Refs. 21Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Crossref PubMed Scopus (489) Google Scholar, 27Lloyd A.J. Thomann H.-U. Ibba M. Söll D. Nucleic Acids Res. 1995; 23: 2886-2892Crossref PubMed Scopus (45) Google Scholar. Synthesis of lipid precursors was confirmed by TLC on silica and by negative ion ES-MS as in Ref. 19Breukink E. van Heusden H.E. Vollmerhaus P.J. Swiezewska E. Brunner L. Walker S. Heck A.J.R. Kruijff de B. J. Biol. Chem. 2003; 278: 19898-19903Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar. Four tRNASer genes, tRNASer(1), tRNASer(2), tRNASer(3), and tRNASer(4) corresponding to (anticodon/locus tag) GCU/SP2253, UGA/SP2258, UGA/SP2291, and GGA/SP2247, respectively, and four UGC anticodon tRNAAla genes (1-4 with locus tags SP2270, SP2282, SP2295, and SP2243, respectively) were identified in the S. pneumoniae TIGR4 genome (26Tettelin H. Nelson K.E. Paulsen I.T. Eisen J.A. Read T.D. Peterson S. Heidelberg J. DeBoy R.T. Haft D.H. Dodson R.J. Durkin S. Gwinn M. Kolonay J.F. Nelson W.C. Peterson J.D. Umayam L.A. White O. Salzberg S.L. Lewis M.R. Radune D. Holtzapple E. Khouri H. Wolf A.M. Utterback T.R. Hansen C.L. McDonald L.A. Feldblyum T.V. Angiuoli S. Dickinson T. Hickey E.K. Holt I.E. Loftus B.J. Yang F. Smith H.O. Venter J.C. Dougherty B.A. Morrison D.A. Hollingshead S.K. Fraser C.M. Science. 2001; 293: 498-506Crossref PubMed Scopus (1119) Google Scholar). Of these, tRNAAla(2)UGC and tRNAAla(3)UGC were located within blocks of sequence that were identical for 3.294 kb 5′ and 1.995 kb 3′ to the gene of interest, and were not amenable to PCR amplification. For the remaining genes, primers 12-23 were designed starting at 250 bp upstream and 250 bp downstream of the mature tRNA sequence (supplemental Table 1), and genes encoding Pn16 tRNAAla(1), Pn16 tRNAAla(4), Pn16 tRNASer(1-4), and 159 tRNAAla(1) were amplified with TaqDNA polymerase; 159 tRNAAla(4) and 159 tRNASer(2) were amplified by platinum Pfx DNA polymerase, and 159 tRNASer(4) was amplified with PWO DNA polymerase. All products were of the expected size (0.6 kb) and were sequenced. No conditions could be found for the amplification of 159 tRNASer(3). 159 tRNASer(1) was amplified with platinum Pfx DNA polymerase; however, most unexpectedly, a clean 2-kb product was obtained, the 3′ termini of which were tagged with ATP and TA cloned into a linearized pCR®2.1 vector, according to the manufacturer's instructions (Invitrogen). The 2-kb insert was then sequenced using the vector-specific m13 primer sequences (supplemental Table 1) either side of the insert. Techniques employed to isolate, preparatively acylate, and assay tRNAAla and tRNASer are described in supplemental Materials and Methods. This assay followed the cycling of tRNASer between MurM and SerRS. To a 0.2-ml assay was added 50 mm HEPES, 30 mm KCl, 10 mm MgCl2, pH 7.6, 1 mm DTT, 1.5% (w/v) CHAPS, 0.25 mm NADH, 2 mm phosphoenolpyruvate, 0.2 mm ATP, 6.2 mg·ml-1 total tRNA159 (1.5 μm in terms of tRNASer159), 10 mm l-serine, 4.89 μm SerRS, 52.5 μmol·min-1·ml-1 myokinase, 6.60 μmol·min-1·ml-1 pyruvate kinase, 10.50 μmol·min-1·ml -1 lactate dehydrogenase, and 0.14 μm MurM159. The ΔA340 of NADH (ϵ340 nm = 6220 m-1·cm-1) was followed at 37 °C, and MurM159 activity was then initiated with 25 μm lipid II. These assays were designed to follow the transfer of label from [3H]alanyl-tRNAAla and [3H]seryl-tRNASer to the peptidoglycan precursor. Initial experiments examining the stability of the aminoacyl linkage to the tRNA suggested that at 37 °C at the pH employed in the spectrophotometric method (7.6) the half-life of M. flavus [3H]alanyl-tRNAAla was 9.8 min. However, this could be extended to 46 min by dropping the pH of the assay to 6.8.3 Therefore, to avoid interference by depletion of acyl-tRNA substrate in MurM assays, initial rate data were usually obtained at pH 6.8 within the first 10 min of reaction, where loss of alanyl-tRNAAla through chemical deacylation was <5%. Transfer of 3H-Amino-acid between [3H]Acyl-tRNA and Lipid-linked Peptidoglycan Precursors—To follow generation of 3H-alanylated lipid precursors, an assay mix typically in a final volume of 35 μl routinely contained 50 mm MOPS, 30 mm KCl, 10 mm MgCl2, pH 6.8, 1.5% (w/v) CHAPS, 1 mm DTT, 1 mm l-alanine, 10 μm lipid substrate, and MurM (as indicated). Reactions were initiated by (unless otherwise indicated) the addition of 0.45 μm [3H]alanyl-tRNAAla (800-1000 cpm·pmol-1 unless stated otherwise) and were incubated at 37 °C for times specified in the text (although initial rate data were usually taken from the first 2 min of reaction). Reactions were terminated at the appropriate time by the addition of 35 μl of ice-cold 6 m pyridinium acetate, pH 4.5, and 70 μl of ice-cold butan-1-ol. The incubations were rapidly mixed and centrifuged for 5 min at 1 °C at 13,000 × g, after which the butan-1-ol phase was washed with 70 μl of water and then assayed for 3H by liquid scintillation counting. To follow generation of 3H-serylated lipid precursors, exactly the same procedure was followed, except that the 1 mm l-alanine and 0.45 μm [3H]alanyl-tRNAAla were replaced with 1 mm l-serine and 0.45 μm [3H]seryl-tRNASer. Transfer of 3H-Amino-acid between [3H]Acyl-tRNA and UDP-linked Peptidoglycan Precursors—To follow generation of 3H-alanylated UDP-MurNAc peptide precursors, an assay mix to follow the MurM-catalyzed UDP-MurNAc-peptide-dependent loss of label from M. flavus [3H]alanyl-tRNAAla was devised. In a final volume of 80 μl, assays contained 50 mm MOPS, 30 mm KCl, 10 mm MgCl2, pH 6.8, 1.5% (w/v) CHAPS, 1 mm DTT, 1 mm l-alanine, UDP-MurNAc peptide substrate (as indicated in the text), and 0.58 μm MurM159. Reactions were initiated by 0.45 μm [3H]alanyl-tRNAAla and were incubated at 37 °C where 10-μl samples were taken up to 3 min. Remaining [3H]alanyl-tRNAAla was quantitated by trichloroacetic acid precipitation as described in supplemental Materials and Methods relative to control incubations carried out without MurM or UDP-MurNAc peptide. Nonlinear regression analyses of dependences of MurM initial velocity on substrate concentration were performed using GraphPad Prism 4 software. To gain insight into the activity of MurM in vivo, we examined the peptide structure of muramidase digests of Pn16 and 159 peptidoglycan by reverse-phase HPLC (Fig. 2). This revealed that the major cross-linked species in Pn16 peptidoglycan is dimeric, composed of two adjacent peptides, without any dipeptide branch between them (peak 4). Unbranched single peptide specie" @default.
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• W2017007825 title "Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan" @default.
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• W2017007825 doi "https://doi.org/10.1074/jbc.m708105200" @default.
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