The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum
2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês
10.1074/jbc.m807175200
ISSN1083-351X
AutoresMaría Fiuza, Marc J. Canova, Delphine Patin, Michal Letek, Isabelle Zanella‐Cléon, Michel Becchi, Luís M. Mateos, Dominique Mengin‐Lecreulx, Virginie Molle, José A. Gil,
Tópico(s)Biofuel production and bioconversion
ResumoThe Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. Due to the increasing number of antibiotic-resistant strains and the emergence of new pathogenic microorganisms, one of the biggest challenges for modern biomedical research is the continuous development of new antimicrobial drugs targeting bacterial essential mechanisms such as cell division or peptidoglycan (PG) 4The abbreviations used are: PGpeptidoglycanMurNAcN-acetylmuramic acidSTPKSer/Thr protein kinaseGSTglutathione S-transferaseMSmass spectrometryMS/MStandem MSLCliquid chromatographyESIelectrospray ionizationTEVtobacco etch virus biosynthesis (1Vicente M. Hodgson J. Massidda O. Tonjum T. Henriques-Normark B. Ron E.Z. FEMS Microbiol. Rev. 2006; 30: 841-852Crossref PubMed Scopus (66) Google Scholar). The bacterial cell wall PG is a giant molecule that sustains the shape of the bacterial cell and contains the outward forces generated in maintaining an osmotic pressure gradient against the environment. Without this PG layer the cell integrity would be ruptured, and this could lead to cell death. Therefore the PG biosynthesis machinery represents a promising source of putative targets for antibacterial chemotherapy (2Barbosa M.D. Ross H.O. Hillman M.C. Meade R.P. Kurilla M.G. Pompliano D.L. Anal. Biochem. 2002; 306: 17-22Crossref PubMed Scopus (22) Google Scholar, 3Barreteau H. Kovac A. Boniface A. Sova M. Gobec S. Blanot D. FEMS Microbiol. Rev. 2008; 32: 168-207Crossref PubMed Scopus (494) Google Scholar). peptidoglycan N-acetylmuramic acid Ser/Thr protein kinase glutathione S-transferase mass spectrometry tandem MS liquid chromatography electrospray ionization tobacco etch virus The biosynthesis of bacterial PG is a complex two-stage process (4van Heijenoort J. Press A. Escherichia Coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, DC1996: 1025-1034Google Scholar). The first stage involves the assembly of the disaccharide peptide monomer unit by enzymes located in the cytoplasm or at the inner surface of the cytoplasmic membrane (3Barreteau H. Kovac A. Boniface A. Sova M. Gobec S. Blanot D. FEMS Microbiol. Rev. 2008; 32: 168-207Crossref PubMed Scopus (494) Google Scholar, 5van Heijenoort J. Nat. Prod. Rep. 2001; 18: 503-519Crossref PubMed Scopus (359) Google Scholar). The peptide moiety of the monomer unit is assembled stepwise by the successive additions of l-alanine, d-glutamic acid, meso-diaminopimelic acid or l-lysine, and d-alanyl-d-alanine to UDP-N-acetylmuramic acid (UDP-MurNAc). These steps are catalyzed by specific peptide synthetases (ligases), which are designated as MurC, MurD, MurE, and MurF, respectively, all participating in non-ribosomal peptide bond formation with the concomitant hydrolysis of ATP. The MurNAc-pentapeptide motif of the resulting nucleotide precursor is then transferred by the MraY translocase onto the undecaprenyl phosphate carrier molecule, generating the lipid intermediate I. The subsequent addition of the N-acetylglucosamine motif of UDP-GlcNAc onto lipid I generates lipid II in a reaction catalyzed by MurG (6Mengin-Lecreulx D. Texier L. Rousseau M. van Heijenoort J. J. Bacteriol. 1991; 173: 4625-4636Crossref PubMed Google Scholar). The second stage of the PG biosynthesis consists in the polymerization by transglycosylation and transpeptidation reactions of the disaccharide pentapeptide monomers, a reaction taking place in the periplasmic space and that is catalyzed by the penicillin-binding proteins. The PG biosynthesis pathway enzymes, which are essential and specific for bacteria, represent important potential targets for screening novel antibacterial compounds. Due to the growing emergence of bacterial multiresistance to currently used antibiotics, the discovery of new therapeutic compounds has indeed become a necessity. In recent years, an extensive search for specific inhibitors interfering with the cytoplasmic steps of this pathway, and in particular with the four steps catalyzed by the Mur ligases, has been developed (2Barbosa M.D. Ross H.O. Hillman M.C. Meade R.P. Kurilla M.G. Pompliano D.L. Anal. Biochem. 2002; 306: 17-22Crossref PubMed Scopus (22) Google Scholar, 7Kotnik M. Anderluh P.S. Prezelj A. Curr. Pharm. Des. 2007; 13: 2283-2309Crossref PubMed Scopus (64) Google Scholar, 8El Zoeiby A. Sanschagrin F. Levesque R.C. Mol. Microbiol. 2003; 47: 1-12Crossref PubMed Scopus (274) Google Scholar). The UDP-Mur-NAc:l-alanine ligase (MurC), encoded by the murC gene, represents such an interesting candidate for drug development (9Ehmann D.E. Demeritt J.E. Hull K.G. Fisher S.L. Biochim. Biophys. Acta. 2004; 1698: 167-174Crossref PubMed Scopus (31) Google Scholar, 10Zawadzke L.E. Norcia M. Desbonnet C.R. Wang H. Freeman-Cook K. Dougherty T.J. Assay Drug Dev. Technol. 2008; 6: 95-103Crossref PubMed Scopus (20) Google Scholar). Recently, different phosphinic acid derivatives and substrate analogues have been identified as Mur ligase inhibitors (11Strancar K. Blanot D. Gobec S. Bioorg. Med. Chem. Lett. 2006; 16: 343-348Crossref PubMed Scopus (50) Google Scholar, 12Strancar K. Boniface A. Blanot D. Gobec S. Arch. Pharm. (Weinheim). 2007; 340: 127-134Crossref PubMed Scopus (28) Google Scholar). Corynebacterium glutamicum is a rod-shaped non-pathogenic Gram-positive actinomycete widely used in the industrial production of amino acids such as l-lysine and l-glutamic acid (13Hermann T. J. Biotechnol. 2003; 104: 155-172Crossref PubMed Scopus (511) Google Scholar). C. glutamicum has been extensively studied as a model microorganism due to the strategies employed by this actinomycete to achieve a rod-shaped morphology. In fact, the mechanisms taking place in C. glutamicum happened to be completely different from that of Escherichia coli or Bacillus subtilis (14Daniel R.A. Errington J. Cell. 2003; 113: 767-776Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 15Letek M. Ordonez E. Vaquera J. Margolin W. Flardh K. Mateos L.M. Gil J.A. J. Bacteriol. 2008; 190: 3283-3292Crossref PubMed Scopus (104) Google Scholar), whereas the number of genes involved in cell division and PG biosynthesis in C. glutamicum is lower (16Letek M. Fiuza M. Ordonez E. Villadangos A.F. Ramos A. Mateos L.M. Gil J.A. Antonie Van Leeuwenhoek. 2008; 94: 99-109Crossref PubMed Scopus (49) Google Scholar). Interestingly, an earlier work on the phosphoproteome of C. glutamicum (17Bendt A.K. Burkovski A. Schaffer S. Bott M. Farwick M. Hermann T. Proteomics. 2003; 3: 1637-1646Crossref PubMed Scopus (136) Google Scholar) identified MurC has being phosphorylated in vivo, suggesting that protein phosphorylation plays a much broader function in C. glutamicum than was previously expected. Recently, we described the characterization of the four STPKs from C. glutamicum ATCC 13869 and highlighted their role in cell division (18Fiuza M. Canova M.J. Zanella-Cleon I. Becchi M. Cozzone A.J. Mateos L.M. Kremer L. Gil J.A. Molle V. J. Biol. Chem. 2008; 283: 18099-18112Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Moreover, Thakur and Chakraborti (19Thakur M. Chakraborti P.K. Biochem. J. 2008; 415: 27-33Crossref PubMed Scopus (54) Google Scholar) showed that MurD from Mycobacterium tuberculosis was phosphorylated by the Ser/Thr protein kinase (STPK) PknA, although no further characterization of the role of the phosphorylation on the MurD enzyme activity was investigated. Therefore, it was tempting to speculate that MurC in C. glutamicum could also be regulated by STPK phosphorylation. The focus of this work is to study the regulation of MurC in C. glutamicum via phosphorylation. As a first step in deciphering the potential role/participation of the corynebacterial STPKs in the regulation of MurC activity, we confirmed its specific phosphorylation by the PknA kinase through a combination of in vitro phosphorylation assays and mass spectrometric identification of the different MurC phosphorylation sites. Moreover, we demonstrated that the murein ligase activity of MurC was negatively regulated upon its phosphorylation. To our knowledge, this work represents the first evidence of a Mur enzyme regulated by phosphorylation. Bacterial Strains and Growth Conditions—Bacterial strains and plasmids are described in Table 1. Strains used for cloning and expression of recombinant proteins were E. coli TOP10 (Invitrogen) and E. coli BL21(DE3)Star (Stratagene), respectively. E. coli cells were grown and maintained at 37 °C in LB medium supplemented with 100 μg/ml ampicillin and/or 50 μg/ml kanamycin, when required. The murC temperature-sensitive E. coli strain H1119 was grown at 30 °C in 2YT (1.6% Bactotrypton, 1.0% Bactoyeast extract, 0.5% NaCl, pH 7.0) medium and was used for genetic complementation experiments with plasmids carrying wild-type or mutated copies of the murC gene. C. glutamicum cells were grown at 30 °C in TSB (Trypticase soy broth, Oxoid) or TSA (TSB containing 2% agar) medium supplemented with 12.5 μg/ml kanamycin. Plasmids to be transferred by conjugation from E. coli to corynebacteria were introduced by transformation into the donor strain E. coli S17-1. Mobilization of plasmids from E. coli S17-1 to C. glutamicum R31 was accomplished as described previously (20Mateos L.M. Schafer A. Kalinowski J. Martin J.F. Puhler A. J. Bacteriol. 1996; 178: 5768-5775Crossref PubMed Google Scholar).TABLE 1Bacterial strains and plasmids used in this studyStrains or plasmidsGenotype or descriptionSource or referenceE. coli TOP10F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG; used for general cloningInvitrogenE. coli BL21 (DE3) StarF2 ompT hsdSB(rB2 mB2) gal dcm (DE3); used to express recombinant proteins in E. coliStratageneE. coli H1119murC temperature-sensitive mutant(23)E. coli S17-1Mobilizing donor strain, pro recA, which possesses an RP4 derivative integrated into the chromosome(38)C. glutamicum 13869Wild-type control strainATCCC. glutamicum R31C. glutamicum ATCC 13869; derivative used as recipient in conjugation experiments(39)pETTevpET15b (Novagen) derivative including the replacement of the thrombin site coding sequence with a tobacco etch virus (TEV) protease site(40)pTEVmurCpTEV derivative used to express His-tagged fusion of MurCThis workpGEX4T-3E. coli vector designed to make GST gene fusionsGE HealthcarepGEXApGEX4T-3 derivative used to express GST fusion of PknA cytoplasmic domain(18)pGEXBpGEX4T-3 derivative used to express GST fusion of PknB cytoplasmic domain(18)pGEXLpGEX4T-3 derivative used to express GST fusion of PknL cytoplasmic domain(18)pTEVGfullpETTev derivative used to express His-tagged PknG(18)pEDivMobilizable plasmid able to replicate in E. coli and C. glutamicum; kan and cat resistance genesA. Ramos (unpublished)pEDivmurHispEDiv derivative used to express His-tagged fusion of MurC in C. glutamicumThis workpGEXpknAT179ApGEXA derivative used to express GST fusion of PknA cytoplasmic domain carrying the mutation T179AThis workpGEXpknAT181ApGEXA derivative used to express GST fusion of PknA cytoplasmic domain carrying the mutation T181AThis workpGEXpknAT179/181ApGEXA derivative used to express GST fusion of PknA cytoplasmic domain carrying the mutations T179A and T181AThis workpGEXpknAK49ApGEXA derivative used to express GST fusion of PknA cytoplasmic domain carrying the mutation K49AThis workpTEVmurC1TpTEVmurC derivative used to express His-tagged fusion of MurC1T carrying the mutation T362AThis workpTEVmurC2TpTEVmurC1T derivative used to express His-tagged fusion of MurC2T carrying the mutation T362A/T365AThis workpTEVmurC3TpTEVmurC2T derivative used to express His-tagged fusion of MurC3T carrying the mutation T362A/T365A/T51AThis workpTEVmurC4TpTEVmurC3T derivative used to express His-tagged fusion of MurC4T carrying the mutation T362A/T365A/T51A/T120AThis workpTEVmurC5TpTEVmurC4T derivative used to express His-tagged fusion of MurC5T carrying the mutation T362A/T365A/T51A/T120A/T167AThis workpTEVmurC6TpTEVmurC5T derivative used to express His-tagged fusion of MurC6T carrying the mutation T362A/T365A/T51A/T120A/T167A/T133AThis workpTEVmurCT51ApTEVmurC derivative used to express His-tagged fusion of MurCT51A carrying the mutation T51AThis workpTEVmurCT120ApTEVmurC derivative used to express His-tagged fusion of MurCT120A carrying the mutation T120AThis workpTEVmurCT133ApTEVmurC derivative used to express His-tagged fusion of MurCT133A carrying the mutation T133AThis workpTEVmurCT167ApTEVmurC derivative used to express His-tagged fusion of MurCT167A carrying the mutation T167AThis workpTEVmurCT362ApTEVmurC derivative used to express His-tagged fusion of MurCT362A carrying the mutation T362AThis workpTEVmurCT365ApTEVmurC derivative used to express His-tagged fusion of MurCT365A carrying the mutation T365AThis workpTrc99AE. coli vector allowing high level expression under the IPTG inducible trc promoterPharmaciapTrc99murCpTrc99A derivative carrying murCThis workpTrc99murC1TpTrc99A derivative carrying murC1T (T362A)This workpTrc99murC2TpTrc99A derivative carrying murC2T (T362A/T365A)This workpTrc99murC3TpTrc99A derivative carrying murC3T (T362A/T365A/T51A)This workpTrc99murC4TpTrc99A derivative carrying murC4T (T362A/T365A/T51A/T120A)This workpTrc99murC5TpTrc99A derivative carrying murC5T (T362A/T365A/T51A/T120A/T167A)This workpTrc99murC6TpTrc99A derivative carrying murC6T (T362A/T365A/T51A/T120A/T167A/T133A)This work Open table in a new tab Cloning, Expression, and Purification of MurC Proteins—First, the murC gene was cloned to generate a recombinant MurC protein expressed in E. coli. Therefore, the murC gene was amplified by PCR using C. glutamicum ATCC 13869 genomic DNA as a template and the primers pair murC1/murC2 (Table 2), containing NdeI and NheI restriction sites, respectively. The 1461-bp amplified product was digested by NdeI and NheI and ligated to the pETTev vector (Table 1) generating the pTEVmurC plasmid. E. coli BL21(DE3)Star cells transformed with this construction were used for expression and purification of His6-tagged MurC, as previously described (21Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Finally, the purified His6-tagged MurC was treated with TEV protease according to the manufacturer's instructions (Invitrogen). Secondly, overexpression and purification of MurC from C. glutamicum cultures was performed using standard PCR strategies. The murC gene from C. glutamicum was amplified using the primers pair murC1/murHisNdeI2 (Table 2). The PCR product carrying a His tag at its C-terminal end was digested with NdeI and subsequently cloned under the control of the Pdiv promoter into plasmid pEDiv (Table 1). The resulting expression vector, named pEDivmurHis, was introduced by conjugation into C. glutamicum R31. Purification of the soluble His6-tagged MurC protein from C. glutamicum was performed as described previously (21Molle V. Brown A.K. Besra G.S. Cozzone A.J. Kremer L. J. Biol. Chem. 2006; 281: 30094-30103Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).TABLE 2Primers used in this studyPrimerGene5′ to 3′ SequenceaRestriction sites are underlined and specified by brackets.bMutagenized codons are shown in bold.murC1murCGGAATTCCATATGGTGACCACTCCACAC (NdeI)murC2murCTGGAATTCGCTAGCCTAATTGTTTTGCAGCTGATCC (NheI)murHisNdeImurCGGAATTCCATATGCTAATGATGATGATGATGATGATTGTTTTGCAGC (NdeI)N-pknAK49MpknAGATCGCGAAGTAGCCATCATGGTACTGCGCCCGGAATTTTCCC-pknAK49MpknAGGAAAATTCCGGGCGCAGTACCATGATGGCTACTTCGCGATCN-pknAT179ApknAGCCGCTGCTGTGCCTTTGGCCCGCACCGGCATGGTGGTGC-pknAT179ApknACACCACCATGCCGGTGCGGGCCAAAGGCACAGCAGCGGCN-pknAT181ApknAGCTGTGCCTTTGACCCGCGCCGGCATGGTGGTGGGTACTC-pknAT181ApknAAGTACCCACCACCATGCCGGCGCGGGTCAAAGGCACAGCN-pknAT179A/T181ApknAGCCGCTGCTGTGCCTTTGGCCCGCGCCGGCATGGTGGTGGGTC-pknAT179A/T181ApknAACCCACCACCATGCCGGCGCGGGCCAAAGGCACAGCAGCGGCN-murC362murCGATTACGCACACCACCCAGCGGAAGTAACTGCAGTGCTCC-murC362murCGAGCACTGCAGTTACTTCCGCTGGGTGGTGTGCGTAATCN-murC362/365murCGCACACCACCCAGCGGAAGTAGCTGCAGTGCTCAGCGCTGCGC-murC362/365murCCGCAGCGCTGAGCACTGCAGCTACTTCCGCTGGGTGGTGTGCN-murC365murCCACCACCCAACGGAAGTAGCTGCAGTGCTCAGCGCGGCGC-murC365murCCGCAGCGCTGAGCACTGCAGCTACTTCCGTTGGGTGGTGN-murC51murCGATGCCAAAGATTCCCGCGCCTTGCTTCCACTCCGCGCCC-murC51murCGGCGCGGAGTGGAAGCAAGGCGCGGGAATCTTTGGCATCN-murC120murCGAATTGCTGGAAGGCTCCGCCCAGGTCTTGATCGCGGGTC-murC120murCACCCGCGATCAAGACCTGGGCGGAGCCTTCCAGCAATTCN-murC167murCACCAATGCGCACCATGGAGCTGGTGAGGTCTTTATCGCTC-murC167murCAGCGATAAAGACCTCACCAGCTCCATGGTGCGCATTGGTN-murC133murCACCCACGGTAAGACCTCCGCCACCTCTATGTCTGTGGTAC-murC133murCTACCACAGACATAGAGGTGGCGGAGGTCTTACCGTGGGTmurH1119-1murCTTTAATCATGACCACTCCACACTTGG (BspHI)murH1119-2murCCTTACAGATCTCTAATTGTTTTGCAGCTG (BglII)a Restriction sites are underlined and specified by brackets.b Mutagenized codons are shown in bold. Open table in a new tab In Vitro Kinase Assays—In vitro phosphorylation was performed with 2 μg of MurC in 20 μl of buffer P (25 mm Tris-HCl, pH 7.0, 1 mm dithiothreitol, 5 mm MgCl2, 1 mm EDTA) with 200 μCi/ml [γ-33P]ATP corresponding to 65 nm (PerkinElmer Life Sciences, 3000 Ci/mmol), and 0.5 μg of kinase. Plasmids pGEXA, pGEXB, pGEXL, and pTEVGfull (Table 1) were used for the expression and purification in E. coli of the four recombinant STPKs from C. glutamicum as previously described (18Fiuza M. Canova M.J. Zanella-Cleon I. Becchi M. Cozzone A.J. Mateos L.M. Kremer L. Gil J.A. Molle V. J. Biol. Chem. 2008; 283: 18099-18112Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). After 15-min incubation, the reaction was stopped by adding sample buffer and heating the mixture at 100 °C for 5 min. The reaction mixtures were analyzed by SDS-PAGE. After electrophoresis, gels were soaked in 20% trichloroacetic acid for 10 min at 90 °C, stained with Coomassie Blue, and dried. Radioactive proteins were visualized by autoradiography using direct exposure films. Analysis of the Phosphoamino Acid Content of Proteins—MurC sample (5 μg) phosphorylated in vitro by the GST-tagged PknA, and unreacted [γ-33P]ATP were separated by one-dimensional gel electrophoresis and electroblotted onto an Immobilon polyvinylidene difluoride membrane. The 33P-labeled protein bands were detected by autoradiography and excised from the Immobilon blot and hydrolyzed in 6 m HCl for 1 h at 110 °C. The acid-stable phosphoamino acids released were separated by electrophoresis in the first dimension at pH 1.9 (800 V/h) in 7.8% acetic acid, 2.5% formic acid, followed by ascending chromatography in the second dimension in 2-methyl-1-propanol/formic acid/water (8:3:4, v/v). After migration, radioactive compounds were detected by autoradiography. Authentic phosphoserine, phosphothreonine and phosphotyrosine were run in parallel and visualized by staining with ninhydrin. Cloning and Purification of PknA Mutant Proteins—Site-directed mutagenesis was directly performed on the pGEXA expression plasmid (Table 1) using inverse-PCR amplification with the following self-complementary primers (Table 2): N-pknAT179A/C-pknAT179A, N-pknAT181A/C-pknAT181A, N-pknAT179A-T181A/C-pknAT179A-T181A, and N-pknAK49M/C-pknAK49M to generate pGEXpknAT179A, pGEXpknAT181A, pGEXpknAT179A/T181A, and pGEXpknAK49M, respectively (Table 1). All constructs were verified by DNA sequencing. The different GST-tagged recombinant fusion proteins were overexpressed and purified as reported earlier (18Fiuza M. Canova M.J. Zanella-Cleon I. Becchi M. Cozzone A.J. Mateos L.M. Kremer L. Gil J.A. Molle V. J. Biol. Chem. 2008; 283: 18099-18112Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Site-directed Mutagenesis—The six threonine residues from C. glutamicum MurC identified by mass spectrometry after in vitro phosphorylation with GST-tagged PknA were replaced by alanine residues by site-directed mutagenesis using inverse-PCR amplification. A first PCR was carried out using pTEVmurC (Table 1) as a template with the primers pair N-murC362 and C-murC362 (Table 2) to generate pTEVmurC1T (T362A). A second PCR was carried out using pTEVmurC (Table 1) as a template with the primers pair N-murC362/365 and C-murC362/365 (Table 2) to generate pTEVmurC2T (T362A/T365A). This mutant and the subsequent additional mutants were used as templates in subsequent PCR reactions using the following primers pairs: N-murC51 and C-murC51, N-murC120 and C-murC120, N-murC167 and C-murC167, and N-murC133 and C-murC133 (see Table 2) to generate pTEVmurC3T (T362A/T365A/T51A), pTEVmurC4T (T362A/T365A/T51A/T120A), pTEVmurC5T (T362A/T365A/T51A/T120A/T167A), and pTEVmurC6T (T362A/T365A/T51A/T120A/T167A/T133A), respectively. Individual mutants were generated using pTEVmurC (Table 1) as a template with the primers pairs N-murC51/C-murC51, N-murC120/C-murC120, N-murC133/C-murC133, N-murC167/C-mur-C167, N-murC362/C-murC362, and N-murC365/C-murC365 (Table 2) to generate pTEVmurCT51A, pTEVmurCT120A, pTEVmurCT133A, pTEVmurCT167A, pTEVmurCT362A, and pTEVmurC T365A, respectively (Table 1). All the resulting constructs were verified by DNA sequencing. The different His6-tagged mutant proteins were overexpressed and purified, as described above. MS Analysis—Purified wild-type and mutant MurC proteins were subjected to in vitro phosphorylation by GST-tagged PknA as described above, excepted that [γ-33P]ATP was replaced with 5 mm ATP. Subsequent analyses using NanoLC/nanospray/tandem mass spectrometry (LC-ESI/MS/MS) were performed as previously described (18Fiuza M. Canova M.J. Zanella-Cleon I. Becchi M. Cozzone A.J. Mateos L.M. Kremer L. Gil J.A. Molle V. J. Biol. Chem. 2008; 283: 18099-18112Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Immunoblotting—Corynebacterial MurC purified either from E. coli or C. glutamicum was loaded on a 10% polyacrylamide gel, electrophoresed, blotted on polyvinylidene difluoride, and detected using either monoclonal mouse anti-phospho-threonine, -serine, -tyrosine, or polyclonal rabbit anti-His antibodies used at 1:100 and 1:10,000 dilution, respectively. Alkaline phosphatase-conjugated anti-mouse or anti-rabbit was used as a secondary antibody at a 1:5,000 dilution. Complementation with C. glutamicum MurC—Plasmids pTEVmurC, pTEVmurC1T, pTEVmurC2T, pTEVmurC3T, pTEVmurC4T, pTEVmurC5T, and pTEVmurC6T were used as templates for PCR amplification of the murC gene with the primers pair murH1119-1/murH1119-2 (Table 2) containing a BspHI and BglII restriction site, respectively. The resulting 1461-bp products, carrying the wild-type and mutated gene copies, respectively, were digested with BspHI and BglII, and cloned into the plasmid vector pTrc99A (22Amann E. Ochs B. Abel K.J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (878) Google Scholar) between the compatible NcoI and BamHI sites, generating pTrcmurC, pTrcmurC1T, pTrcmurC2T, pTrcmurC3T, pTrcmurC4T, pTrcmurC5T, and pTrcmurC6T, respectively. These plasmids allowing high level expression of the C. glutamicum MurC and MurC mutants under the control of the strong isopropyl 1-thio-β-d-galactopyranoside-inducible trc promoter, were transformed in the E. coli MurC temperature-sensitive mutant strain H1119 (23Lugtenberg E.J. v Schijndel-van Dam A. J. Bacteriol. 1972; 110: 35-40Crossref PubMed Google Scholar). Transformants were grown at the permissive temperature (30 °C) before being shifted to the restrictive temperature (42 °C). Complementation was judged by the ability of the mutant to grow at the restrictive temperature. UDP-MurNAc l-alanine Ligase Assay—The l-alanine-adding activity of wild-type and mutant MurC proteins was assayed according to Liger et al. (24Liger D. Masson A. Blanot D. van Heijenoort J. Parquet C. Eur. J. Biochem. 1995; 230: 80-87Crossref PubMed Scopus (58) Google Scholar) by following the formation of UDP-MurNAc-l-[14C]alanine in 40 μl of reaction mixture containing 100 mm Tris-HCl, pH 8.6, 20 mm MgCl2, 20 mm ammonium sulfate, 0.5 mm l-[14C]alanine (0.6 KBq, 5.5 Gbq/mmol, Amersham), 1 mm UDP-MurNAc, 5 mm ATP, and enzyme (25 μl of an appropriate dilution in buffer A: 20 mm potassium phosphate, pH 7.2, containing 1 mm dithiothreitol and 10% glycerol). The mixture was incubated at 37 °C for 30 min and the reaction was stopped by addition of 8 μl of acetic acid, followed by lyophilization. To test the effect of the phosphorylation of MurC on its enzyme activity, the assay was performed as described above excepted that the MurC enzyme (20 μg/ml) was preincubated overnight at 37 °C with or without wild-type PknA (16 μg/ml), or with PknA_K49M mutant (30 μg/ml) in buffer A supplemented with 2 mm ATP and 5 mm MgCl2. The radioactive product UDP-MurNAc-l-[14C]alanine and substrate l-[14C]alanine were separated by reversed-phase high-performance liquid chromatography on a Nucleosil 5C18 column (4.6 × 150 mm, Alltech France) using 50 mm ammonium formate, pH 3.9, as the eluent, at a flow rate of 0.6 ml/min. Detection was performed with a radioactive flow detector (model LB506-C1, Berthold France, La Garenne-Colombes, France) using the Quicksafe Flow 2 scintillator (Zinsser Analytic, Maidenhead, UK) at 0.6 ml/min. Quantitation was carried out with the Radiostar software (Berthold). MurC Is a Substrate of the C. glutamicum PknA—The peptide moiety of the PG monomer unit is assembled stepwise in the cytoplasm by the successive actions of four Mur ligases designated as MurC, MurD, MurE, and MurF. These enzymes share limited sequence identity but have several highly conserved regions that map primarily to the active site. Each of them comprises three structural domains: an N-terminal domain with a Rossmann-type fold primarily responsible for binding of the UDP-MurNAc(-peptide) substrate, a large central ATP-binding (ATPase) domain, and a C-terminal domain associated with binding of the amino acid substrate (25Bouhss A. Mengin-Lecreulx D. Blanot D. van Heijenoort J. Parquet C. Biochemistry. 1997; 36: 11556-11563Crossref PubMed Scopus (85) Google Scholar). Based on protein sequence alignments and on the three-dimensional structure of the E. coli MurC protein (26Deva T. Baker E.N. Squire C.J. Smith C.A. Acta Crystallogr D. Biol. Crystallogr. 2006; 62: 1466-1474Crossref PubMed Scopus (32) Google Scholar), these three domains in the C. glutamicum MurC may extend between Met1-Gly118, Ser119-Arg334, and Arg335-Asn486, respectively (Figs. 1 and 4). By comparing the amino acid sequences of the MurC, -D, -E, and -F ligases from different bacterial genera, several conserved residues were identified. This analysis led to the characterization of the ATP-bind
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