Purification and biochemical characterization of Mur ligases from Staphylococcus aureus

Biochimie 92 (2010) 1793e1800

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Research paper

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus Delphine Patin a, b, Audrey Boniface a,1, Andreja Kova
c c, Mireille Hervé a, b, Sébastien Dementin a, 2, Hélène Barreteau a, Dominique Mengin-Lecreulx a, b, Didier Blanot a, b, * a b c

Univ Paris-Sud, Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, Orsay F-91405, France CNRS, Orsay F-91405, France Fakulteta za Farmacijo, A
sker
ceva 7, Univerza v Ljubljani, 1000 Ljubljana, Slovenia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 May 2010 Accepted 15 July 2010 Available online 24 July 2010

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-Nacetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His6-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted L-Ala, L-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for L-Ala. S. aureus MurE was very specific for L-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and L-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (L-lysine and mesodiaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively mesodiaminopimelic acid and L-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis. Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Staphylococcus aureus Peptidoglycan Mur ligases Escherichia coli

1. Introduction Infections caused by multidrug-resistant Gram-positive bacteria represent a major public health burden in terms of morbidity, mortality and health care costs [1]. Staphylococcus aureus is the

Abbreviations: A2pm, diaminopimelic acid; HPLC, high-performance liquid chromatography; iGln, isoglutamine; MRSA, methicillin-resistant Staphylococcus aureus; MurNAc, N-acetylmuramic acid; Ni2þ-NTA, Ni2þ-nitrilotriacetate; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin-layer chromatography; Sa, Staphylococcus aureus origin of a gene or an enzyme; Ec, Escherichia coli origin of a gene or an enzyme. * Corresponding author. Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, Bâtiment 430, Université Paris-Sud 11, 91405 Orsay, France. Tel.: þ33 1 69 15 81 65; fax: þ33 1 69 85 37 15. E-mail address: [email protected] (D. Blanot). 1 Present address: Institut Pasteur, Unité des Membranes Bactériennes, Département de Microbiologie, 25-28, rue du Dr. Roux, 75724 Paris Cedex 15, France. 2 Present address: CNRS, Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, 31, chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France. 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.07.009

aetiological agent for a wide range of human infections, including abscesses, septicaemia, arthritis and endocarditis. The increased prevalence of methicillin-resistant (MRSA) and vancomycininsensitive S. aureus strains, and the emergence of communityacquired MRSA, make investigations into the pathogenicity of this species imperative [2]. Inevitably, this focuses research into the development of novel antimicrobial agents, which requires a rigorous study of staphylococcal metabolism and physiology [3]. Peptidoglycan is an essential component of bacterial cell-wall [4]. Its main function is to preserve cell integrity by withstanding the inner osmotic pressure. It is composed of long glycan chains cross-linked by short peptides. Its biosynthesis is a complex process which constitutes a target for new antibacterial compounds. The assembly of the peptide moiety of peptidoglycan [5] involves a superfamily of enzymes, the Mur ligases [6e8]. These enzymes have three substrates: i) ATP; ii) a nucleotide precursor consisting of UDP-N-acetylmuramic acid (UDP-MurNAc) possibly linked to an amino acid or a peptide; iii) an amino acid or a dipeptide. They share a common reaction mechanism [9] and similar 3D structures [10]. In Escherichia coli, they catalyze the successive additions to UDP-MurNAc of L-Ala (MurC), D-Glu (MurD),

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meso-diaminopimelic acid (meso-A2pm) (MurE), and D-Ala-D-Ala (MurF), thereby leading to the synthesis of the cytoplasmic precursor UDP-MurNAc-L-Ala-g-D-Glu-meso-A2pm-D-Ala-D-Ala. Within the bacterial world, there exists a restricted variability for the amino acid added by MurC (L-Ala, Gly, L-Ser) and the dipeptide added by MurF (D-Ala-X, where X is D-Ala, D-lactate or D-Ser). In contrast, the nature of the amino acid (generally a diaminoacid) added by MurE varies greatly according to the species: meso-A2pm in most Gram-negative bacteria, mycobacteria and bacilli, L-Lys in most Gram-positive bacteria, L-Orn in spirochetes; in rare cases, meso-lanthionine, LL-A2pm, D-Lys or L-homoserine, can even be found [4,5,11]. It is noteworthy that the amino acid present at position 2 or 3 of the peptide stem of mature peptidoglycan sometimes differs from the amino acid substrate of MurD or MurE: for instance, Disoglutamine (D-iGln) or threo-hydroxy-3-glutamic acid are present at position 2 instead of D-Glu in the peptidoglycan of some species [4,11]. These residues originate from modifications (amidation, hydroxylation, etc.) occurring after the action of the Mur ligases, generally at the level of lipid II. As far as position 2 is concerned, the amino acid substrate of MurD is D-Glu in all of the bacterial species examined to date. The properties and substrate specificity of the Mur ligases have been well studied in E. coli. It is important to extend this study to the enzymes of other bacteria, in particular S. aureus. Preliminary work has been done a few decades ago with crude extracts or partially purified preparations [12e18]. More recently, purifications to homogeneity of staphylococcal MurC and MurD have been reported [19,20]. In this paper, we describe the purification and characterization of the four Mur ligases from S. aureus, which catalyze the synthesis of the cytoplasmic precursor UDP-MurNAc-LAla-g-D-Glu-L-Lys-D-Ala-D-Ala. The kinetic parameters for natural and alternate substrates are reported. These properties are compared to those of the E. coli Mur ligases. Moreover, a comparison of the specific activities of the ligases determined in a crude extract of S. aureus to those of the purified enzymes is made, which allows us to estimate the number of molecules of these enzymes present in cells and to make a correlation between enzyme levels and cell requirements for peptidoglycan synthesis.

Amersham Biosciences, and pTrcHis60 and pET2160 plasmids for expression of proteins with a C-terminal 6histidine (His6) tag have been previously described [26,27]. The pMAK705 plasmid [28] was a kind gift of S. R. Kushner and the BW25113 strain and the pKD3 and pKD46 plasmids used for gene disruption experiments [29] were kindly provided by B. Wanner via the E. coli Genetic Stock Center (Yale University, New Haven). pTrc99A-derivative plasmids pAM1005, pABD16, pMLD117 and pMLD116 expressing the E. coli murC, murD, murE and murF genes, respectively, have been previously described [7,30e32]. Unless otherwise noted, 2 YT medium [33] was used for growing cells, and growth was monitored at 600 nm with a Shimadzu UV-1601 spectrophotometer. When required, cultures were supplemented with ampicillin, kanamycin, and chloramphenicol (100, 50 and 25 mg ml1, respectively). The temperature-sensitive E. coli mutant strains H1119 (murC), TKL-11 (murE) and TKL46 (murF), used for functional complementation experiments, were obtained from the Phabagen collection [34,35]. The murD temperature-sensitive E. coli strain BWTsmurD was constructed as follows. The murD gene was deleted and replaced by a chloramphenicol resistance cartridge in the chromosome of strain BW25113 by using the procedure of Datsenko and Wanner [29] and oligonucleotides Inact1-murD and Inact2-murD (Supplementary Table S1). As the murD gene is essential, the inactivation procedure was applied to cells harbouring a plasmid, pABD16 [7], expressing a wild-type copy of murD gene. The murD::CmR mutation was then transduced by phage P1 into the BW25113 strain carrying the pMAKmurD plasmid (see Section 2.4) that bears a thermosensitive replicon, generating the strain BWTsmurD that could grow at 30  C but not at 42  C. The H1119 and BWTsmurD strains were grown in 2 YT medium, the TKL-11 strain in low salt LB medium (0.2% NaCl) supplemented with thymine (100 mg ml1), and the TKL46 strain in no salt LB medium supplemented with thymine (100 mg ml1). 2.3. General DNA techniques and E. coli cell transformation Plasmid purification kits were purchased from MachereyeNagel. Standard procedures for molecular biology were used [36]. E. coli cells were made competent and transformed with plasmid DNA according to Dagert and Ehrlich [37], or by electroporation.

2. Materials and methods 2.4. Construction of plasmids 2.1. Materials DNA restriction enzymes and synthetic oligonucleotides were purchased from New England Biolabs and Eurofins-MWG, respectively. The nucleotide precursors UDP-MurNAc, UDP-MurNAc-L-Ala, UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ala-g-D-Glu-L-Lys and UDP-MurNAc-L-Ala-g-D-Glu-meso-A2pm were prepared according to Babi
c et al. [21]. L-[14C]Ala (5.99 GBq mmol1) and L-[14C]Ser (6.07 GBq mmol1) were purchased from Perkin Elmer, [14C]Gly (3.88 GBq mmol1) from CEA, and D-[14C]Glu (2.03 GBq mmol1) from American Radiolabeled Chemicals, Inc. The other radiolabelled substrates, UDP-[14C]MurNAc, UDP-MurNAc-L-Ala-D-[14C] Glu, UDP-MurNAc-L-Ala-g-D-[14C]Glu-L-Lys, and D-Ala-D-[14C]Ala, were prepared according to published procedures [9,22e24]. 2.2. Bacterial strains and growth conditions E. coli strain DH5a (Invitrogen) was used as a host for plasmids. Strain C43(DE3) (Avidis) was used for the overproduction of MurCSa, MurDSa and MurFSa, and strain BL21(DE3)pLysS (Novagen) for that of MurESa. The pREP4groESL plasmid allowing overproduction of the bacterial chaperones was obtained from K. Amrein [25]. The plasmid vector pTrc99A was obtained from

The S. aureus mur genes were amplified from the S. aureus chromosome (strain 8325) by PCR using appropriate primers (Supplementary Table S1). The amplified products were cloned into the pET2160 vector (T7 promoter), generating plasmids that encode proteins with a His6-tag at the C-terminal extremity (further details are available in Supplementary material). All the constructions were verified by DNA sequencing (Eurofins-MWG). For overproduction of Mur ligases, plasmids pET2160::murCSa, pET2160::murDSa and pET2160::murFSa were used and transformed into E. coli C43(DE3), and plasmid pET2160::murESa into E. coli BL21(DE3) pLysS harboring plasmid pREP4groESL. For complementation assays, the murC, murD, murE and murF genes were cloned into the expression vector pTrcHis60, generating pTrcHis60::murCSa, pTrcHis60::murDSa, pTrcHis60::murESa and pTrcHis60::murFSa. A plasmid allowing conditional (thermosensitive) expression of E. coli murD was constructed as follows: the E. coli murD gene and its proximal region was amplified by PCR using oligonucleotides 5’EcmurD-BamHI and 3’EcmurD-SacI (Supplementary Table S1) and the resulting DNA fragment was cut with BamHI and SacI and cloned between the same sites of the low-copy number plasmid vector pMAK705, whose replication is impaired at 42  C, generating pMAKmurD.

D. Patin et al. / Biochimie 92 (2010) 1793e1800

2.5. Complementation of thermosensitive E. coli mutants The different pTrcHis60-derivative plasmids allowing expression of the S. aureus mur genes were tested for complementation of the E. coli thermosensitive mutants H1119, BWTsmurD, TKL-11 and TKL46, that exhibit defective expressions of the murC, murD, murE and murF genes (and/or altered activities of the corresponding proteins), respectively, when grown at 42  C. In each case, the transformants were plated on two plates, one being incubated at the permissive temperature (30  C) and the other at the restrictive temperature (42  C), and complementation was judged by the ability of the mutants to grow at the restrictive temperature. The empty vector pTrcHis60 was used as a negative control, and plasmids pAM1005 [32], pABD16 [7], pMLD117 [31], and pMLD116 [30], that express the E. coli murC, murD, murE and murF genes, respectively, were used as positive controls in these assays. 2.6. Overproduction and purification of S. aureus Mur enzymes Overnight precultures of E. coli C43(DE3) harbouring either pET2160::murCSa, pET2160::murDSa or pET2160::murFSa, were used to inoculate 2 YT medium supplemented with ampicillin (1-litre cultures). The cultures were incubated at 37  C with shaking until the optical density at 600 nm reached 0.8. Isopropyl b-Dthiogalactopyranoside (IPTG) was added at a final concentration of 1 mM, and incubation was continued for 3 h at 37  C. In the case of MurE, an overnight preculture of E. coli BL21(DE3) pLysS harbouring both plasmids pREP4groESL and pET2160::murESa was used to inoculate 2 YTeampicillinekanamycinechloramphenicol medium. The culture was incubated with shaking at 37  C. When the optical density reached 0.9, the temperature of the culture was decreased to 22  C and IPTG was added at a 100 mM final concentration. Growth was continued for 16 h at 22  C. In all cases, cells were harvested at 4  C and the pellet was washed with cold 20 mM phosphate buffer, pH 7.2, containing 1 mM dithiothreitol (buffer A). Bacteria were resuspended in buffer A (6 ml) and disrupted by sonication in the cold using a Bioblock Vibracell 72412 sonicator. The resulting suspension was centrifuged at 4  C for 30 min at 200,000g with a Beckman TL100 apparatus, and the pellet was discarded. The supernatant was kept at 20  C. The His6-tagged proteins were purified on Ni2þ-nitrilotriacetate 2þ (Ni -NTA)-agarose following the manufacturer’s recommendations (Qiagen). All procedures were performed at 4  C. The supernatant was mixed for 1 h with the polymer previously washed with buffer A containing 0.3 M KCl and 10 mM imidazole. Washing and elution steps were performed with a discontinuous gradient of imidazole (20e400 mM) in buffer A containing 0.3 M KCl. Protein contents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and relevant fractions were pooled and dialyzed against 100 volumes of buffer A. Glycerol (10% final concentration) was added for storage of the proteins at 20  C. Protein concentrations were determined by quantitative amino acid analysis with a Hitachi L8800 analyzer (ScienceTec) after hydrolysis of samples in 6 M HCl at 105  C for 24 h. 2.7. Determination of the kinetic constants The activity assays for the S. aureus Mur ligases measured the formation of radioactive product from a radioactive substrate using reaction mixtures (final volume, 50 ml) containing: MurC: 100 mM TriseHCl, pH 8.6, 5 mM MgCl2, 2.5 mM DTT, 20 mM ammonium sulfate, ATP, UDP-MurNAc and L-Ala (varying concentrations), and enzyme (25 ml of an appropriate dilution in buffer A).

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MurD: 100 mM TriseHCl, pH 8.8, 15 mM MgCl2, ATP, UDPMurNAc-L-Ala and D-Glu (varying concentrations), and enzyme (25 ml of an appropriate dilution in buffer A). MurE: 100 mM TriseHCl, pH 8.6, 15 mM MgCl2, ATP, UDPMurNAc-L-Ala-D-Glu and L-Lys (varying concentrations), and enzyme (15 ml of an appropriate dilution in buffer A). MurF: 100 mM TriseHCl, pH 8.6, 20 mM MgCl2, ATP, UDP-MurNAcL-Ala-g-D-Glu-L-Lys and D-Ala-D-Ala (varying concentrations), and enzyme (25 ml of an appropriate dilution in buffer A). In all cases, the mixtures were incubated for 30 min at 37  C, and the reaction was terminated by the addition of glacial acetic acid (10 ml) followed by lyophilization. Generally, the radiolabelled amino acid or dipeptide was used. In that case, radioactive substrate and product were separated by thin-layer chromatography (TLC) on silica gel plates LK6D (Whatman) using 1-propanol/ammonium hydroxide/water (6:3:1; v/v) as the mobile phase, and the radioactive spots were located and quantified with a radioactivity scanner (model MultiTracemaster LB285, Berthold). However, when the nucleotide precursor concentration was much lower than that of the amino acid or dipeptide owing to great difference in their respective Km values, the radiolabelled nucleotide was used. Radioactive substrate and product were then separated by HPLC on a Nucleosil 100C18 5 U column (150  4.6 mm; Alltech France) using 50 mM ammonium formate, pH 3.2 (MurC) or 3.9 (MurD), 50 mM sodium phosphate and 7.2 mM sodium hexanephosphonate, pH 2.5/acetonitrile (98.5:1.5, v/v) (MurE) [22], or 50 mM ammonium acetate, pH 6.0 (MurF), at a flow rate of 0.6 ml min1. Radioactivity was detected with a flow detector (model LB506-C1, Berthold) using the Quicksafe Flow 2 scintillator (Zinsser Analytic) at 0.6 ml min1. Quantification was performed with the Radiostar software (Berthold). In all cases, the enzyme concentration was chosen so that substrate consumption was <20%, the linearity being ensured within this interval even at the lowest substrate concentration. Data were fitted to the equation v ¼ VmaxS/(Km þ S) by the LevenbergeMarquardt method [38], and values  standard deviations at 95% of confidence were calculated. The MDFitt software developed by M. Desmadril (UMR 8619 CNRS, Orsay, France) was used for this purpose. 2.8. Preparation of S. aureus crude extracts and determination of the Mur ligase specific activities S. aureus RN4220 cells (800 ml cultures) were grown in 2 YT medium at 37  C with shaking. When the optical density reached 0.9, cells were harvested at 4  C, washed with cold buffer A, resuspended in 12 ml of the same buffer and disrupted with a FastPrep Instrument (4 cycles of 15 s at maximum speed, with 0.1e0.11 mm glass beads). The resulting suspension was centrifuged for 30 min at 90,000g and the supernatant was dialyzed against buffer A and stored at 20  C. This preparation, designated as the crude soluble extract, contained 25 mg of proteins (as determined by quantitative amino acid analysis). The specific activities of the four Mur ligases in this extract were measured using the conditions described in Section 2.7, with radiolabelled amino acid or dipeptide and saturating concentrations of the substrates [in MurC and MurD assays, Mg2þ was used at 20 mM and 30 mM, respectively (see Section 3.3)]. 3. Results 3.1. Functional complementation The murC, murD, murE and murF genes of S. aureus were cloned into the pTrcHis60 vector, generating pTrcHis60c::murCSa,

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pTrcHis60::murDSa, pTrcHis60::murESa and pTrcHis60::murFSa plasmids, respectively, in which gene expression is under the control of the strong IPTG-inducible trc promoter. These plasmids were tested for functional complementation of lytic thermosensitive E. coli mutant strains having defects in the expression of the orthologue mur genes. In the cases of MurC, MurD and MurF, growth of transformants was observed at both 30  C and 42  C, indicating that expression of the staphylococcal ligases perfectly complemented the specific defects of the corresponding E. coli mutants. Complementation did not require IPTG induction, thereby indicating that basal expression of the enzymes from the plasmids was sufficient to restore cell viability. No complementation of the different E. coli mutants was observed with the empty pTrcHis60 vector. The situation was different in the case of MurE, where no complementation of the E. coli murE mutant could be observed by the pTrcHis60::murESa plasmid. This finding was not surprising, however, because as described in Section 3.3, E. coli and S. aureus MurE orthologue enzymes exhibit completely different substrate specificities that could not allow inter-species complementation. As a matter of fact, we previously showed that over-expression of the S. aureus murE gene in E. coli cells resulted in the replacement of meso-A2pm by L-lysine in the peptidoglycan of the latter species, and ultimately to cell lysis [39].

3.2. Protein expression and purification Staphylococcal Mur ligases were overproduced in E. coli as C-terminal His6-tagged forms, using pET2160 derivative plasmid constructs (see Section 2.4). When IPTG induction (1 mM) was performed for 3 h at 37  C, MurC, MurD and MurF proteins were significantly overproduced and mainly recovered in the soluble fraction of cell extracts, as judged by SDS-PAGE analysis (data not shown). Under the same conditions, MurE was shown to aggregate and was essentially detected as inclusion bodies. To solve this problem, the relevant E. coli strain was transformed by plasmid pREP4groESL, which allows the expression of chaperone proteins [25], and the culture was performed at 22  C instead of 37  C. However, under these new conditions, over-expression of the murE gene led to cell lysis, as already observed [39] (Fig. 1). We found that lowering the IPTG concentration to 100 mM avoided this toxicity

Fig. 1. Growth of E. coli BL21(DE3)/pLysS/pREP4groESL/pET2160::murESa. Cells were grown at 22  C without IPTG (dots), or with 50 mM (triangles), 100 mM (diamonds) or 500 mM (squares) IPTG, added at t ¼ 0.

effect (Fig. 1). With these precautions, MurE could be recovered mainly in the soluble fraction. The proteins were purified in native form on Ni2þ-NTA-agarose, using elution with an imidazole gradient. The yields were quite good, about 20, 32, 11, and 39 mg l1 of culture for MurC, MurD, MurE and MurF, respectively. The proteins were homogenous by SDS-PAGE (Fig. 2) and the m/z values determined by MALDI-TOF mass spectrometry were in agreement with their calculated molecular masses. A post-translational removal of the N-terminal methionine residue apparently occurred in the case of MurF (Supplementary Table S2). 3.3. Enzymatic characterization In order to define appropriate assay conditions for the purified enzymes, optimal pH and magnesium concentration values were determined (Table 1). pH-activity profiles displayed bell shapes with broad optima around 8.6. This seems to be a characteristic of all Mur ligases, which are active within the 8e9.2 pH range [40]. In contrast, dose-response analyses for magnesium yielded sharp curves and a different optimal concentration for each enzyme, between 5 and 20 mM. The same had been observed with the E. coli orthologues (from 5 mM for MurD to 100 mM for MurE) [32,41e43]. Except noted otherwise, the optimal values for pH and magnesium mentioned in Section 2.7 were used. The kinetic parameters of MurCSa are shown in Table 2. The Vmax value is higher than that determined by Kurokawa et al. [19], and lower than the Vmax for the E. coli enzyme. The Km values for UDPMurNAc and L-Ala are similar to those determined by the Japanese group. As far as ATP is concerned, a strong inhibition over 4 mM was seen when the enzyme was used at the optimal Mg2þ concentration of 5 mM (Table 1). When the Mg2þ concentration was raised to 20 mM, where the enzyme kept 57% of its activity, this phenomenon disappeared, thereby indicating that it was due to the binding of MurC with free ATP. The Km value (2.0 mM) for ATP found in these conditions was much higher than that of Kurokawa et al. (0.1 mM) [19]. The reason for this discrepancy is unclear; we can only notice the narrow range of ATP concentration used by these authors (0.05e0.5 mM) in contrast to ours (0.1e10 mM). As mentioned in Section 1, L-Ser or Gly can replace L-Ala as the amino acid substrate of MurC in some species. In E. coli, where L-Ala is the amino acid present at position 1 of the peptide stem, L-Ser and Gly can also be used as substrates by MurC in vitro, but at 4.4and 13-fold lower rates, respectively [44]. The same trend was seen

Fig. 2. SDS-PAGE of purified staphylococcal Mur ligases (2 mg each). MW, molecular mass standards (kDa). Staining was performed with Coomassie brilliant blue R250.

D. Patin et al. / Biochimie 92 (2010) 1793e1800 Table 1 Optimal pH and magnesium concentration values for the staphylococcal Mur ligases. Parameter

MurC

pH MgCl2 (mM)

8.2e8.8 5

MurD

MurE

8.4e9.6 15

8.4e9.2 15

MurF 8.4e9.0 20

for MurCSa: at fixed concentrations of the substrates (4 mM ATP, 2.5 mM UDP-MurNAc, 1.5 mM amino acid), the activities with L-Ala, 1 L-Ser and Gly were 1770, 410 and 250 nmol min mg1, respectively. Therefore, L-Ser and Gly were incorporated 4.3- and 7-fold less rapidly, respectively, than L-Ala, thereby explaining, at least partially, why essentially the latter amino acid is present at position 1 in mature peptidoglycan [4,11]. The kinetic parameters of MurDSa are shown in Table 3. While the Vmax is higher than that for MurDEc, the Km values for the three substrates are higher; consequently, the efficiency of the staphylococcal enzyme is not very different from that of E. coli. Similarly to MurC, a strong inhibition by excess ATP, observed at the optimal Mg2þ concentration (15 mM), was not retrieved at a higher concentration (30 mM), where the enzyme retained 80% of its activity. Here again, the Km value for ATP was high (5.4 mM). As a matter of fact, the Km values found in this study were totally different from those determined by Walsh et al. [20] (Table 3). The assay conditions used by these authors greatly differ from ours: pH, temperature, and ATP concentration range were 8.0, 25  C, and 0e1 mM, respectively, instead of 8.8, 37  C, and 0.2e10 mM in our case. These differences perhaps explain the discrepancy between their Km values and ours. In the mature peptidoglycan of S. aureus, D-iGln is present at position 2 of the peptide. Although the amide function is known to be added at a later stage of biosynthesis [45], we wanted to check whether D-iGln could be a substrate for MurDSa as well. D-iGln and radioactive UDP-MurNAc-L-Ala were incubated with a high concentration of enzyme. The HPLC profile of the assay displayed a new peak which was assigned to UDP-MurNAc-L-Ala-D-iGln (data not shown). However, the corresponding activity (17  0.7 nmol min1 mg1), which was 2200-fold lower than that determined for D-Glu in the same conditions, showed that this in vitro phenomenon has no physiological relevance. As already stated, MurE adds the amino acid at position 3 of the peptide stem. This amino acid is L-Lys in S. aureus and meso-A2pm in E. coli. While the substrate specificity of MurE has been well studied in E. coli [42,46e48], to the best of our knowledge it has never been studied in S. aureus with the pure enzyme. The kinetic parameters of purified MurESa are shown in Table 4 and are compared with

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Table 3 Kinetic parameters of MurD ligases from S. aureus and E. coli. Parametera

MurDSab 1

1

MurDSac

MurDEcd

Vmax (mmol min mg ) 35  6 32  3.7 8.4 5.4  1.9e 0.084  0.010 0.14 Km, ATP (mM) 0.041  0.020 0.29  0.034 0.0075 Km, UMA (mM) 0.13  0.044 0.53  0.025 0.055 Km, D-Glu (mM) 1 mg1 mM1) 6.5  2.5e 380  63 60 Vmax/KATP m (mmol min Vmax/KUMA (mmol min1 mg1 mM1) 1400  740 110  18 1100 m 60  7.5 150 Vmax/KDm-Glu (mmol min1 mg1 mM1) 450  170 e

a

UMA, UDP-MurNAc-L-Ala. This study. The concentrations of the fixed substrates were 2 mM for ATP, 0.75 mM for UMA, and 1 mM for D-Glu. The concentration ranges for the varied substrates were 0.2e10 mM for ATP, 0.01e0.75 mM for UMA, and 0.02e1 mM for D-Glu. c Ref. [20]. d Refs. [43,60]. e In order to avoid inhibition by free ATP, these values were determined at 30 mM 2þ Mg . b

those of the E. coli enzyme. Similarly to MurD, the Vmax is higher for the staphylococcal MurE orthologue. The Km values for ATP and UDP-MurNAc-dipeptide are similar for both enzymes. The specificity for the amino acid substrate was studied (Table 5). The S. aureus enzyme is devoid of meso-A2pm-adding activity, whereas that of E. coli possesses a very weak L-Lys-adding activity (6000-fold lower Vmax/Km ratio as compared to meso-A2pm). Contrary to the E. coli enzyme, MurESa accepts L-ornithine as substrate, but the efficiency is 400-fold lower than that with L-Lys. The fourth Mur ligase, MurF, adds the preformed dipeptide DAla-D-Ala to UDP-MurNAc-tripeptide. Owing to the respective specificities of the MurE enzymes, the nucleotide precursor contains at position 3 L-Lys in S. aureus and meso-A2pm in E. coli. The kinetic parameters of both MurF enzymes are shown in Table 6. While the maximum velocity was higher for the staphylococcal enzyme, the Km values for the three substrates were similar. Contrary to the E. coli enzyme [49], no inhibition of MurFSa by UDPMurNAc-tripeptide was observed. Interestingly, MurFSa could indifferently use L-Lys- and meso-A2pm-containing forms of the nucleotide precursor, with the same efficiency: at fixed substrates concentrations (5 mM ATP, 400 mM of nucleotide precursor and DAla-D-Ala), the observed activities were 54  3 and 58  2 mmol min1 mg1, respectively. This characteristic is shared by the E. coli enzyme [49], revealing that the discrimination for the amino acid residue at position 3 is entirely supported by MurE in both species.

Table 2 Kinetic parameters of MurC ligases from S. aureus and E. coli. Parametera

MurCSab 1

1

Vmax (mmol min mg ) Km, ATP (mM) Km, UM (mM) Km, L-Ala (mM) 1 mg1 mM1) Vmax/KATP m (mmol min 1 mg1 mM1) Vmax/KUM m (mmol min Vmax/KLm-Ala (mmol min1 mg1 mM1) a

2.9 2.0 0.28 0.44 1.4 10 6.4

      

e

0.2 0.3e 0.02 0.13 0.24e 1.0 1.9

MurCSac

MurCEcd

0.66 0.10 0.24 0.44 6.6 2.7 1.5

17.3 0.45 0.10 0.020 38 170 860

UM, UDP-MurNAc. This study. The concentrations of the fixed substrates were 4 mM for ATP, 2 mM for UM, and 2 mM for L-Ala. The concentration ranges for the varied substrates were 0.1e10 mM for ATP, 0.025e3 mM for UM, and 0.05e6 mM for L-Ala. c Ref. [19]. d Ref. [32]. e In order to avoid inhibition by free ATP, these values were determined at 20 mM 2þ Mg . b

Table 4 Kinetic parameters of MurE ligases from S. aureus and E. coli. Parametera

MurESab

Vmax (mmol min1 mg1) Km, ATP (mM) Km, UMAG (mM) Km, AA (mM) 1 mg1 mM1) Vmax/KATP m (mmol min (mmol min1 mg1 mM1) Vmax/KUMAG m 1 Vmax/KAA mg1 mM1) m (mmol min

5.3 0.53 0.087 0.55 10 61 9.6

      

MurEEcc 0.3 0.10 0.022 0.26 2 16 4.6

1.4 0.62 0.080 0.040 2.3 17 35

a UMAG, UDP-MurNAc-L-Ala-D-Glu. The amino acid substrate (AA) was L-Lys for MurESa and meso-A2pm for MurEEc. b This study. The concentrations of the fixed substrates were 5 mM for ATP, 0.3 mM for UMAG, and 5 mM for L-Lys. The concentration ranges for the varied substrates were 0.5e10 mM for ATP, 0.02e0.5 mM for UMAG, and 1e20 mM for L-Lys. c Ref. [47].

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D. Patin et al. / Biochimie 92 (2010) 1793e1800

Table 5 Alternate substrates for MurE ligases from S. aureus and E. coli. MurESaa

Substrate

L-Lys

meso-A2pm L-Orn

MurEEcb

Vmax (mmol min1 mg1)

Km (mM)

Vmax/Km (mmol min1 mg1 mM1)

Vmax (mmol min1 mg1)

Km (mM)

Vmax/Km (mmol min1 mg1 mM1)

5.3  0.3 nac 0.87  0.12

0.55  0.26 na 37  8

9.6  4.6 na 0.023  0.006

0.07 1.4 na

11 0.040 na

0.0064 35 na

a This study. The concentrations of the fixed substrates were 5 mM for ATP and 0.3 mM for UMAG. The concentration ranges for the varied substrates were 1e20 mM for LLys, and 1e25 mM for L-Orn. b Ref. [47]. c No detectable activity with 5 mg enzyme.

3.4. Specific activities of Mur ligases in crude extracts from S. aureus In order to correlate the in vitro activities of the Mur ligases with the in vivo requirements for cell-wall peptidoglycan synthesis, the levels of the four enzymes were determined in extracts from exponentially growing S. aureus wild-type cells. The specific activities determined for the MurCSa, MurDSa, MurESa and MurFSa ligases in crude cell extracts from strain RN4220 were 7.5, 19, 17 and 83 nmol min1 mg of protein1, respectively. A comparison of these values to those of the purified enzymes (2900, 35,000, 5300 and 71,000 nmol min1 mg1, respectively; see Tables 2e6) allowed us to estimate the numbers of molecules of the four Mur ligases present in the bacterial cell: about 4100, 850, 4500 and 1800, respectively. 4. Discussion In the present work, the mur genes of S. aureus were cloned and the Mur ligases they encode were overproduced and purified. The staphylococcal murC, murD and murF genes were able to complement the corresponding E. coli thermosensitive strains, thereby reflecting similar specificities of the MurC, MurD and MurF enzymes in S. aureus and E. coli. It was therefore not surprising that the over-expression of these genes in E. coli could be obtained without any problem, yielding these proteins in high amounts (20e40 mg l1 of culture). The case of murE was different. We earlier showed that overexpression of the staphylococcal murE gene in E. coli led to morphological alterations and cell lysis [39]. We demonstrated that this was due to the incorporation of L-Lys, a “wrong” amino acid, into E. coli peptidoglycan, the L-Lys-containing peptide stems not being able to serve as acceptors in transpeptidation reactions. For these reasons, as indeed observed in the present work, no

Table 6 Kinetic parameters of MurF ligases from S. aureus and E. coli. Parametera

MurFSab 1

1

Vmax (mmol min mg ) Km, ATP (mM) Km, UMT (mM) Km, D-Ala-D-Ala (mM) 1 mg1 mM1) Vmax/KATP m (mmol min (mmol min1 mg1 mM1) Vmax/KUMT m Vmax/KDm-Ala-D-Ala (mmol min1 mg1 mM1)

71 0.11 0.046 0.24 640 1800 300

      

MurFEcc 5 0.034 0.016 0.057 200 540 73

16 0.05e0.10 0.07 0.22 160e320 230 73

a UMT, UDP-MurNAc-tripeptide. For MurFSa, the Km values for ATP and D-Ala-DAla were determined with the L-Lys form of UMT; for MurFEc, the meso-A2pm form was used. b This study. The concentrations of the fixed substrates were 5 mM for ATP, 0.5 mM for UMT, and 1.5 mM for D-Ala-D-Ala. The concentration ranges for the varied substrates were 0.05e4 mM for ATP, 0.01e0.6 mM for UMT, and 0.05e5 mM for D-Ala-D-Ala. c Ref. [41].

complementation of the E. coli murE mutant could occur following expression of the S. aureus murE gene. One consequence of this toxicity effect was the difficulty in producing large amounts of purified MurESa. The purification yield (11 mg l1) we obtained for this protein after optimization of expression conditions was, however, quite enough for its biochemical characterization. The availability of the four staphylococcal Mur ligases allowed us to determine the kinetic parameters and to compare them with the E. coli values. In general, the Km values for the substrates were higher for the staphylococcal enzyme. In two cases, MurCSa and MurDSa, the Km for ATP was very high (2 mM); although unusual, such values are not unprecedented (e.g., 3.6 mM for MurE from Thermotoga maritima [22]). Apart from MurCSa, the Vmax values were higher than those of the E. coli enzymes. We also assayed these enzymes for potential alternate substrates. Although it did not exhibit a strict specificity, MurCSa (as well as MurCEc) had a preference for L-Ala, which is the amino acid found at position 1 of staphylococcal (and E. coli) peptidoglycan. On the other hand, the specificity of MurESa is very strict: it hardly accepts L-Orn as a substrate, and not at all meso-A2pm. This specificity mirrors that of the E. coli orthologue, for which L-Lys is a very poor substrate and L-Orn not a substrate at all. It is important to notice that L-Lys, L-Orn, meso-A2pm, and possibly other related amino acids, co-exist in the cytoplasm of bacteria. The strict substrate specificity of the MurE enzymes seems to be necessary to prevent incorporation of “wrong” amino acids into peptidoglycan that brings about deleterious effects [39]. The lack of specificity of the downstream enzymes (MurF, MraY, MurG and glycosyltransferases) for precursors containing a “wrong” amino acid at position 3, as illustrated here for MurF, substantiates this need. The specific activities of the MurCSa, MurDSa, MurESa and MurFSa ligases determined in crude cell extracts of S. aureus were 7.5, 19, 17 and 83 nmol min1 mg of protein1, respectively. Interestingly, these levels were 20- to 40-fold higher than those detected for the orthologue enzymes in E. coli cells [50]. Indeed, specific activities reported for MurDEc, MurEEc and MurFEc in exponentially growing cells of E. coli were 0.72, 0.75 and 2.0 nmol min1 mg of protein1, respectively. It is well known that the peptidoglycan layer of Grampositive bacteria is much thicker than that of Gram-negative species. Recently, the peptidoglycan content of S. aureus cells was precisely determined and shown to be about 10-fold higher than that of E. coli (98 versus 8.5 mmol per g of cell dry weight, expressed in terms of muramic acid content of isolated sacculi) [50,51]. That the Mur ligases are expressed to higher levels in S. aureus cells could therefore be correlated to higher requirements for peptidoglycan synthesis in this bacterial species. The Mur ligases do not seem to be limiting factors participating in the regulation of the flow of metabolites in the pathway for peptidoglycan synthesis. It was earlier shown that their levels in E. coli cells did not vary significantly with growth conditions, suggesting that these enzymes were constitutively expressed at a level in somewhat excess as compared to the cell requirements [50].

D. Patin et al. / Biochimie 92 (2010) 1793e1800

Overexpressing individual Mur ligases genes in E. coli also did not result in an increased rate of formation of peptidoglycan and cellwall thickening. This is likely also the case in S. aureus and other Gram-positive bacteria. In the latter species, however, overproduction of some of the enzymes involved in earlier steps of the pathway were shown to have an impact on the flow of metabolites and cell peptidoglycan content. Recently for instance, overproducing the PEP:UDP-GlcNAc enolpyruvyl transferase MurZ in S. aureus was shown to increase by 20% the cell peptidoglycan content [51]. Furthermore, it was earlier shown that S. aureus mutant strains had developed low-level resistance to vancomycin (VISA strains) by thickening the cell-wall peptidoglycan layer [52], a finding which was then correlated to an increased expression of the fructose-6-phosphate:glutamine amidotransferase GlmS involved in early steps of synthesis of peptidoglycan precursor UDP-GlcNAc [53]. No concomitant variation of the enzymatic activities catalyzing subsequent steps in the pathway, such as the Mur ligases, was reported in these cases, suggesting that these intermediate activities were present in excess and adapted for sustaining increased rates of peptidoglycan synthesis. The recent demonstration that the MurC ligase from Corynebacterium glutamicum could be phosphorylated both in vitro and in vivo by the PknA serine/threonine protein kinase, and that this modification dramatically decreased the enzyme activity [54], suggests that Mur ligase activities could potentially be negatively regulated by such a mechanism, at least in some bacterial species. In conclusion, the present work has allowed the purification and enzymatic study of the four staphylococcal Mur ligases, enzymes which participate in the synthesis of an essential cell-wall polymer and therefore are potential targets for antibacterial agents. The availability of these enzymes and acquired knowledge on their enzymatic properties will be useful for the search for inhibitors, a work that has already started in the case of MurESa [55e59]. Acknowledgments This work was supported by the European Commission through the EUR-INTAFAR project (LSHM-CT-2004-512138), the Centre National de la Recherche Scientifique (PICS 3729), the Ministère de l’Education Nationale, de la Recherche et de la Technologie (scholarship to S.D.), the Délégation Générale pour l’Armement (Contrats Jeune Chercheur 036000104 and 056000030 to A.B.), and the Franco-Slovene Proteus programme.

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