Kinetic Characterization of Lipid II-Ala:Alanyl-tRNA Ligase (MurN) from Streptococcus pneumoniae using Semisynthetic Aminoacyl-lipid II Substrates*

MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (l-Ala/l-Ser) and second (l-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402–6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-l-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is l-Ala-l-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.

The peptidoglycan layer of Streptococcus pneumoniae and other Gram-positive pathogens is cross-linked between Lys at position 3 of its pentapeptide side chain -L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala (2)(3)(4). In Gram-negative organisms, there are direct links between meso-diaminopimelic acid at position 3 and the fourth position D-Ala of a second pentapeptide chain (5). Some Gram-positive bacteria contain direct cross-links between L-lysine and D-alanine, but many Gram-positive bacteria contain a further peptide cross-link comprising one or more amino acids (3,6). The composition of such branched peptidoglycan peptide cross-links varies between bacterial species, as shown in Table 1 (2,7).
The addition of the branched peptide cross-link usually occurs at the stage of lipid intermediate II (although it occurs on UDP-MurNAc-pentapeptide in Weissella viridescens (8)). Residues are added sequentially to the ⑀-amino terminus of L-lysine, in the opposite direction to that of protein synthesis (9 -13). The addition of the amino acid residues of the crosslink is catalyzed by membrane-associated ligases, which utilize aminoacyl-tRNAs as substrates (7,13).
The genetic determinants of branched wall structure in S. pneumoniae are the murM and murN genes (14,15). MurM catalyzes the addition of L-Ala or L-Ser, whereas the addition of the second L-Ala is catalyzed by MurN (16). S. pneumoniae cell walls contain a mixture of directly linked (unbranched) and indirectly linked (branched) peptidoglycan, but the murMN genes are not essential, since direct cross-links can be formed (7,15,17). However, these enzymes do have a role in the phenotype of penicillin resistance, since inactivation of murMN leads to a loss of penicillin resistance (16,17). Clinical strains of penicillin-resistant S. pneumoniae require for the high level resistance phenotype 1) the presence of specific murMN sequences, responsible for dipeptide cross-link formation and 2) specific modified penicillin-binding protein sequences (16 -20). However, certain laboratory S. pneumoniae strains containing resistant murMN alleles do not show penicillin resistance, since they lack high affinity penicillin-binding proteins (35).
The characterization of S. pneumoniae MurM ligases from a highly penicillin-resistant strain (159) and penicillin-susceptible strain (Pn16) has been recently carried out by Lloyd et al. (1), using enzymatically synthesized lipid II substrate (1,(21)(22)(23). The markedly different branching phenotype displayed by S. pneumoniae Pn16 and 159 is rationalized in vitro by the much higher specific activity of MurM 159 over MurM Pn16 with pneumococcal alanyl-tRNA Ala and the higher activity with alanyl-tRNA Ala than with seryl-tRNA Ser (1).
In order to better understand the molecular basis of penicillin resistance caused by MurM and MurN, we wished to kinetically characterize the second ligase MurN in two clinical isolates of S. pneumoniae, one highly penicillin-resistant (159) and the other penicillin-sensitive (Pn16). In order to reconstitute the MurN-catalyzed reaction, we have developed a chemoenzymatic method to prepare the aminoacyl-lipid II substrate for MurN, and we report the specificity of recombinant MurN for lipid II-Ala versus lipid II-Ser substrates.

EXPERIMENTAL PROCEDURES
UDP-MurNAc-pentapeptide Biosynthesis and Purification-Details of preparation and purification of the UDP-MurNAcpentapeptide are reported in Ref. 1.
Synthesis of UDP-MurNAc-hexapeptide (L-Ala)-To 2 ml of 80% (v/v) acetonitrile in water were added 17.2 mg (90 mol) of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 6.9 mg (60 mol) of N-hydroxysuccinimide, 1.9 mg (10 mol) of N-(ethylsulfite)-morpholine, and 7.5 mg (24 mol) of L-alanine-Fmoc. The pH was adjusted to 5.0 if needed. After 20 min of stirring at room temperature, 100 l of 20 mM UDP-MurNAc-pentapeptide (2.3 mg) in 500 mM NaHCO 3 (pH 10.0) were added. The suspension was stirred at room temperature for 3 h, followed by the addition of 100 l of ethanolamine and further incubated for 20 min before the addition of 100 l of piperidine. After 30 min of incubation, 18 ml of H 2 O were added, and the solution was filtered with a nitrocellulose syringe filter (0.20-m pore size). The UDP-MurNAchexapeptide (L-Ala) synthesis was achieved in 66% yield. The filtrate product was freeze-dried and stored at Ϫ20°C.
Purification of UDP-MurNAc-hexapeptides-To isolate UDP-MurNAc-hexapeptide products, the crude filtrates were resuspended in 100 ml of 10 mM ammonium acetate, pH 7.5, and loaded onto a Source 30Q column (26 ϫ 120 mm; Amersham Biosciences) equilibrated in 10 mM ammonium acetate, pH 7.5, and the column was developed with an ammonium acetate gradient from 0 to 300 mM ammonium acetate over 7 column volumes at 15 ml min Ϫ1 . UDP-MurNAc-peptide elution was followed at 254 nm. The UDP-MurNAc-peptide peak was collected, freeze-dried four times to remove trace amounts of the buffer, dissolved in water, and stored at Ϫ20°C. Purification was achieved in 95% yield.
Synthesis of Lipid II-L-Ala and Lipid II-L-Ser-In order to form lipid II hexapeptide substrates, 30 mol of UDP-GlcNAc (Sigma), 2.5 mol of UDP-MurNAc-hexapeptide, 2.5 mol of undecaprenyl phosphate (Larodan Fine Chemicals AB), and 4.5 mg of Micrococcus flavus membranes protein in 0.1 M Tris, pH 8, 5 mM MgCl 2 , 1% (w/v) Triton X-100 in a final volume of 1.5 ml was incubated at 37°C for 3 h. The lipids were extracted and purified by DEAE-cellulose anion exchange chromatography, as described in Ref. 21. Synthesis of lipid precursor was confirmed by TLC on silica and by negative ion electrospray-mass spectrometry as in Ref. 21.
Escherichia coli Strains and Plasmids-Details of E. coli strains and plasmids in this study are indicated in the supplemental materials.
S. pneumoniae Strains and Isolation of Pneumococcal DNA-Details of S. pneumoniae strains and isolation methods of genomic DNA are described in Refs. 19 and 24.
Protein Analytical Methods-SDS-PAGE, protein assay, and Western blotting for histidine tags were performed according to Refs. 25 and 26.
Cloning, Overexpression, and Purification of MurN from S. pneumoniae-The murN genes from S. pneumoniae Pn16 and 159 strains were cloned into the expression vector pBADM-41 to allow expression of MurN fused to a maltose-binding protein (MBP) 2 with N-terminal hexahistidine tag and C-terminal tobacco etch virus (TEV) protease cleavage site (27). The same primers were designed to amplify both alleles (Table S1). The initial start codon of murN was absent in murN-Nco1 Fw primer, and the murN stop codon was present in the MBP-murN-Xho1 Rv primer. The murN genes were amplified from S. pneumoniae Pn16 and 159 genomic DNA using Pfx polymerase (Invitrogen). The primers and conditions are detailed in Table  S1. Amplified products were purified, restricted with NcoI and XhoI, and ligated into similarly restricted pBADM-41 as described in Refs. 26 and 28. Clones carrying the recombinant murN 159 and murN Pn16 genes were verified by sequencing, and one correct clone was retained for expression of each protein (pBADM-41::murN 159 and pBADM-41::murN Pn16 ).
To overexpress the MurN proteins, 1-liter cultures of E. coli TB1, harboring either pBADM-41::murN 159 or pBADM-41::murN Pn16 in LB plus 50 g/ml ampicillin were grown at 37°C to A 600 of 0.7, when murN expression was induced by 0.04% (w/v) L-arabinose. E. coli cells were harvested after 4 h, and the cell pellets were washed at 4°C in 50 mM HEPES, 1 mM MgCl 2 , pH 7.5, and 2 mM ␤-mercaptoethanol. The cells were lysed by sonication on ice and clarified by centrifugation at 10,000 ϫ g. The supernatant was then transferred in fresh tubes and centrifuged at 50,000 ϫ g at 4°C. The subcellular fractions were analyzed by SDS-PAGE, and MBP-MurN 159 and MBP-MurN Pn16 were present in the soluble fraction.

Staphylococcus aureus
Gly-Gly-Gly-Gly-Gly S. pneumoniae L-Ala-L-Ala or L-Ser-L-Ala W. viridescens  1) and were incubated at 37°C for times specified below (although initial rate data were usually taken from the first 4 min of reaction). Reactions were terminated at the appropriate time by the addition of ice-cold 30 l of 6 M pyridinium acetate, pH 4.5, and 60 l of ice-cold n-butyl alcohol. The incubations were rapidly mixed and centrifuged for 5 min at 4°C at 13,000 ϫ g, after which the n-butyl alcohol phase was washed with 70 l of water and then assayed for [ 3 H]scintillation counting in 5 ml of Optiphase scintillation mixture (HiSafeЈ 2 from PerkinElmer Life Sciences). To follow generation of 3 H-serylated lipid precursors, exactly the same procedure was followed, except that the 1 mM alanine and 0.45 M [ 3 H]alanyl-tRNA Ala were replaced with 1 mM serine and 0.45 M [ 3 H]seryl-tRNA Ser (prepared as reported in Ref. 1).
Kinetic Data Analysis-Nonlinear regression analyses of dependences of MurN initial velocity on substrate concentration were performed using GraphPad Prism 4 software.

Preparation of Lipid-linked Cell
Wall Precursors-In order to study the enzymology of MurN, access to its natural substrates (lipid II-L-Ala or lipid II-L-Ser) was required. These substrates were synthesized using a chemoenzymatic route. UDP-MurNAc-L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala was synthesized enzymatically, as described by Lloyd et al. (1), and then chemically coupled with N-terminally protected L-alanine or L-serine to generate UDP-MurNAc-hexapeptide (L-Ala or L-Ser) (Fig. 1), which was then converted to lipid II-L-Ala or lipid II-L-Ser using M. flavus membranes (21).
Synthesis of UDP-MurNAc-hexapeptides-The protocol to attach L-alanine or L-serine on the ⑀-NH 2 of L-lysine of the UDP-MurNAc-L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala ( Fig. 1) was based on a carbodiimide coupling reaction using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) and promoted by N-hydroxysuccinimide (NHS) (29). EDC reacts with a carboxyl group on Fmoc-L-Ala or Fmoc-L-Ser, forming an amine-reactive O-acylisourea intermediate. The addition of NHS forms a water-soluble active ester, which is amine-reactive but more stable than the O-acylisourea EDC adduct, thus increasing the efficiency of EDC-mediated coupling reactions (29). The Fmoc group was then deprotected using piperidine.
To maximize the efficiency of UDP-MurNAc-hexapeptide formation, the pH, the incubation time, the concentrations of coupling reagent, and the protected amino acid were optimized. The activation of the protected amino acid with EDC/ NHS was found to be optimal at pH 5.0, whereas the reaction of the resulting NHS ester with the amine of L-Lys of the UDP-MurNAc-pentapeptide was favored at higher pH, at which the ⑀-NH 2 of L-Lys (pK a 10.3) is more deprotonated. For this reason, the reaction was performed in two stages, first the activation of the protected amino acid was carried at pH 5.0, followed by the addition of UDP-MurNAc-pentapeptide in sodium carbonate at pH 10.0. The coupling reaction time was varied from 30 min up to 24 h, where 3 h has been established to be the optimum time. The same timedependent profile was obtained for the addition of Fmoc-L-Ser to the UDP-MurNAc-pentapeptide.
In order to achieve a good yield, a large excess of coupling reagents and protected amino acid was necessary. It was found that the UDP-MurNAc-pentapeptide must be free of  DECEMBER 12, 2008 • VOLUME 283 • NUMBER 50 ammonium acetate, due to the possible reaction of the ammonia with the activated amino acid. For this reason, some batches of UDP-MurNAc-pentapeptide were further purified by gel filtration. The best results were obtained using 45 eq of EDC, 30 eq of NHS, and 12 eq of protected amino acid with respect to the UDP-MurNAc-pentapeptide, with yields in the range 60 -65%.
The UDP-MurNAc-hexapeptides were analyzed by negative ion electrospray mass spectrometry. Electrospray  (21). Lipid II-L-Ala and lipid II-L-Ser were purified on a DEAE-cellulose column, using the method of Breukink et al. (21), and were analyzed by thin layer chromatography (Fig. 3). The lipid II-L-Ala and lipid II-L-Ser products were analyzed by negative ion electrospray mass spectrometry.   In order to generate native MurN, the MBP fusion proteins were successfully cleaved with TEV protease. However, attempts to separate the cleaved MurN from MBP using affinity chromatography, anion exchange, hydrophobic interaction, or ammonium sulfate precipitation were unsuccessful, suggesting a tight association between MurN and MBP. Possibly  (Table 2). In control experiments without MurN 159 or lipid II-L-Ala, only 3% of the 3 H added to the incubation was accumulated into n-butyl alcohol-extractable products, demonstrating the essential requirement of alanyl-tRNA Ala for MurN activity. Similar results were obtained with lipid II-L-Ser (Table 2). To demonstrate the requirement of MurN for the tRNA portion of the [ 3 H]alanyl-tRNA Ala substrate, complete reactions were treated with 0.1 mg ml Ϫ1 RNase A, which reduced incorporation of 3 H into lipid products to 3%, comparable with control values obtained without lipid II-L-Ala/L-Ser or MurN 159 (Table 2). Similar results were obtained with MurN Pn16 (Table 2). No detectable differences in MurN activity emerged using M. flavus alanyl-tRNAs or S. pneumoniae alanyl-tRNAs. For example, the incorporation percentage of 3 H added to the assay into n-butyl alcoholsoluble material in the MurN Pn16 reaction with lipid II-L-Ala was 82% using S. pneumoniae tRNAs and 80% using M. flavus tRNAs (Table 2)  MurN Substrate Specificity Studies-Felipe et al. (14,16,35) demonstrated that MurN adds an alanine residue to a previously acylated stem peptide. To correlate this in vivo finding with the enzymatic properties of MurN, we assayed MurN activity with lipid II and seryl-tRNA Ser .
In order to determine if lipid II, the substrate of MurM, was also a MurN substrate, the activity of this enzyme was assayed between 0 and 250 M lipid II. The assays were incubated for 6 min at 37°C, using 50 nM MurN 159 and 0.6 M [ 3 H]alanyl-tRNA Ala from M. flavus. No MurN activity was detected using lipid II as a substrate. A time course experiment (from 2 to 120 min, at 200 M lipid II and 50 nM MurN 159) was performed in case the MurN reaction with lipid II was particularly slow. However, even under these conditions, it was not possible to detect any MurN activity with lipid II as substrate. Similar results were obtained with MurN Pn16 . These data were consistent with the in vivo behavior of this enzyme.
MurN was tested with Dependences of MurN 159 and MurN Pn16 on lipid II L-Ala or lipid II L-Ser were hyperbolic and were fitted by nonlinear regression to the Michaelis-Menten equation (Fig. 4). MurN 159 utilized lipid II L-Ala as substrate more efficiently than lipid II L-Ser. Comparison of k cat(app)/ K m(app) for lipid II L-Ala and lipid II L-Ser (Table 3) suggested that the lipid II L-Ser substrate reduced its catalytic efficiency 20-fold. Likewise, MurN Pn16 displayed a preference for lipid II L-Ala as substrate over lipid II L-Ser. The MurN Pn16 catalytic efficiency (k cat(app) /K m(app) ) was 11-fold higher for lipid II L-Ala than lipid II L-Ser (Table 3). No differences in activity were observed between MurN 159 and MurN Pn16 ; the two enzymes were comparable in terms of catalytic efficiency with each substrate (Table 3).

DISCUSSION
MurN is an aminoacyl ligase that adds alanine as the second amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan (14,16). Studies in whole pneumococcal cells suggested that the addition of the dipeptide  The specific activities were calculated from initial rates (10 mM branched lipid at 37°C, methods described under "Experimental Procedures"). The kinetic constants were determined according to the Michaelis-Menten equation. Fitting data was performed by nonlinear regression, using GraphPad Prism 4 software.  DECEMBER 12, 2008 • VOLUME 283 • NUMBER 50 branch does not occur in the cytoplasmic steps of peptidoglycan biosynthesis (as it does in W. viridescens (8,36,37)) but in the lipid-linked stages, indicating that lipid II-L-Ala or lipid II-L-Ser might be the likely substrate for MurN (17). The MurN gene product shares 26% sequence identity with Staphylococcus aureus FemA, which catalyzes the addition of the second and third glycine residues in the branched muropeptide of S. aureus (38,39). Disruption of the femA gene abolishes methicillin resistance in this organism (40). The Fem-ABX proteins have a requirement for aminoacyl-charged tRNAs as substrates for the nonribosomal peptide bond formation of the pentaglycine bridge (40). The functional and sequence similarity of MurN to these proteins suggested that it also uses aminoacyl-tRNAs as substrates.

Enzymology of the Aminoacyl Ligase MurN
We have recently reported the reconstitution and kinetic characterization of MurM from penicillin-resistant and penicillin-sensitive S. pneumoniae (1). The reconstitution and kinetic characterization of both MurM and MurN therefore provides a better understanding of the peptide bridge biosynthesis in S. pneumoniae.
We have developed and optimized for the first time a chemoenzymatic method to prepare the substrates lipid II-L-Ala and lipid II-L-Ser, using a carbodiimide coupling onto the most reactive ⑀-NH 2 group of L-lysine. Using this method, it was possible to prepare 10 -12 mg of UDP-MurNAc-hexapeptide (L-Ala/L-Ser) per reaction with a yield between 40 and 65%. This synthesis is versatile, and other amino acid (glycine) or dipeptides (L-Ala-L-Ala and L-Ser-L-Ala) were also successfully attached on the ⑀-NH 2 of L-lysine of the UDP-MurNAc-pentapeptide (data not shown). The UDP-MurNAc-hexapeptides (L-Ala/L-Ser) were successfully converted into lipid II-L-Ala and lipid II-L-Ser by M. flavus membranes, using the method of Breukink et al. (21). This method could, in principle, be applied to the synthesis of a variety of lipid II-peptide conjugates, which will be very useful to study the enzymology of the FemABX/ MurMN ligase family, and for future work on penicillin-binding proteins from Gram-positive bacteria containing indirect peptidoglycan cross-links.
MurN from S. pneumoniae 159 and Pn16 strains could be expressed as MBP-MurN fusion proteins, but attempts to separate cleaved MurN from MBP were unsuccessful, suggesting a tight association between MurN and MBP. In vivo MurN probably interacts with MurM, as suggested by the work on S. aureus FemX, FemA, and FemB (13,41), and this phenomenon may reflect the high affinity between MurN and MBP. This is the first reconstitution of an aminoacyl-tRNA ligase with a modified lipid II-X substrate, the Fem ABX ligases having been reconstituted together (13). This allows an assessment of kinetic parameters and the substrate specificity of each ligase enzyme. MurN requires an aminoacyl-tRNA substrate for the transfer of L-alanine to lipid II-L-Ala and lipid II-L-Ser, as found by Lloyd Pn16 . This result is in accordance with the cell wall analysis in S. pneumoniae, where only alanine has been found in position 2 of the branched peptide stem (1,14).
It is not clear whether the amino acid selectivity of MurN was due to specific interaction with the tRNA or the amino acid moiety. Recent studies on S. pneumoniae MurM (1) and W. viridescens FemX (8) have shown that these enzymes primarily recognize only the acceptor stem and T⌿C loop of tRNA and that other regions of the tRNA are not required for the peptidyltransferase activity (1,8). In addition, as previously described, MurN uses tRNAs from M. flavus as well as the S. pneumoniae tRNA. These results could lead to the hypothesis that the tRNA is essential for activity but not for amino acid selectivity, but further studies are required.
No significant differences in kinetic parameters were observed between recombinant MurN from S. pneumoniae 159 and Pn16 strains (see Table 3). This result is entirely consistent with the relative absence of polymorphism in the murN gene, which is highly conserved and shows little sequence variation between resistant and susceptible S. pneumoniae strains (MurN 159 and Pn16 differ in only three amino acids: R212Q, E115Q, and S225T). A low level of divergence (between 1 and 2%) also emerged from the comparison of murN genes from clinical isolates and laboratory strains of S. pneumoniae. The absence of polymorphism in the murN gene could also explain the invariable addition of alanine to the second position of the peptide cross-link in S. pneumoniae.
Both MurN enzymes show a kinetic preference for lipid II L-Ala over lipid II L-Ser as substrate: a 20-fold and 11-fold difference in catalytic efficiency for MurN 159 and MurN Pn16 , respectively (Table 3). In the resistant strain 159, this preference matches that of MurM 159 , which is 7-fold more active with alanyl-tRNA Ala than with seryl-tRNA Ser (1). This result therefore rationalizes the peptidoglycan analysis of S. pneumoniae 159 strain, in which the majority of the peptidoglycan is branched and the predominant cross-link is L-Ala-L-Ala (1). In the sensitive strain Pn16, MurM Pn16 is slightly more active with seryl-tRNA Ser (1); however, MurN Pn16 has a somewhat higher catalytic efficiency with lipid II L-Ser as substrate than Mur-M Pn16 . Therefore, it is likely that MurN Pn16 would be able to convert the proportion of lipid II L-Ser generated by MurM Pn16 , which rationalizes the presence of a proportion of L-Ser-L-Ala cross-links in sensitive strains.
The biochemical characterization of MurM and MurN therefore confirms and rationalizes the earlier genetic observations (14 -18), giving a better understanding of the pneumococcal stem-peptide biosynthesis. Ligase MurM is selective for the addition of the first amino acid (alanine or serine) to the lipid II in position 3, and ligase MurN adds only alanine as the second amino acid to lipid II-L-Ala and lipid II-L-Ser. MurM enzymes from penicillin-resistant and -sensitive S. pneumoniae strains have different amino acid selectivity and specific activity; in contrast, the two MurN enzymes do not show any significant kinetic differences. This is in accordance with the presence of extensive sequence polymorphism in the murM gene, which is absent in the murN gene, and the relative abundance of branched peptidoglycan in those strains. In conclusion, MurM is clearly the major determinant in the occurrence and sequence of the dipeptide cross-link, and the role of MurN is to complete the biosynthesis of the dipeptide initiated by MurM.