JOURNAL OF BACTERIOLOGY, Apr. 2006, p. 2528–2532 0021-9193/06/$08.00⫹0 doi:10.1128/JB.188.7.2528–2532.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 7
Kinetic Characterization of the Monofunctional Glycosyltransferase from Staphylococcus aureus Mohammed Terrak and Martine Nguyen-Diste`che* Centre d’Inge´nierie des Prote´ines, Universite´ de Lie`ge, Institut de Chimie, B6a, Sart-Tilman, 4000 Lie`ge, Belgium Received 28 September 2005/Accepted 9 January 2006
The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of ⬃5,800 Mⴚ1 sⴚ1 and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2ⴙ. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site. pentapeptide) as substrate (8, 24). S. aureus MGT was found to be sensitive to moenomycin, whereas E. coli MGT was not. Deletion of the mgt gene in E. coli, H. influenzae, and B. abortus has a slight effect on cell growth in broth culture (5, 19, 24). However, B. abortus cells depleted of MGT were found to be less effective in the initial phase of infection in mice than were wild-type cells (5). Therefore, MGTs seem to play a role in the pathogenicity process of infectious bacteria. Similarly, Pasteurella multocida depleted of class A PBP1c (homologue of E. coli PBP1c) was viable in vitro but showed significant attenuation of pathogenicity in vivo (13). Taken together, these observations suggest that nonessential genes in laboratory conditions and the apparent redundant PBPs involved in PG biosynthesis are probably essential for bacterial development in host environments. S. aureus possesses only one class A PBP (PBP2). In the presence of penicillin, PBP2 can be replaced by the class B low-affinity PBP2a in the methicillin-resistant S. aureus strain (18), suggesting a cooperation between the transpeptidase module of PBP2a and the GT module of class A PBP2. In addition, inactivation by site-directed mutagenesis of the GT activity of PBP2 gives rise to a viable mutant susceptible to methicillin. Glycan chain elongation in the mutant is presumably catalyzed by the MGT (17, 18). This enzyme thus seems to play a role in cell wall assembly. The GTs of E. coli PBP1b, Streptococcus pneumoniae PBP2a, and S. aureus PBP2 have been characterized (3, 9, 21). From site-directed mutagenesis experiments, it was shown that Glu233 in motif 1, which is conserved in all class A PBPs and MGTs, is the key element of the GT catalytic center of E. coli PBP1b (21). Ca2⫹ or Mg2⫹ ions are required for the activity and thus appear to play a catalytic role in the GT reaction (20). Moenomycin and vancomycin derivatives inhibit the GT step
Bacterial wall peptidoglycan (PG) is a net-like macromolecule consisting of glycan strands made of alternating -1,4linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides (22). The final stages of PG polymerization are catalyzed by two sets of membrane-bound penicillin-binding proteins (PBPs) that belong either to class A or class B, depending on the structure and the catalytic activity of their N-terminal module (11). The C-terminal penicillin-binding module of both classes has transpeptidase activity catalyzing peptide cross-linking between adjacent glycan chains (22). In class A, the N-terminal module has glycosyltransferase (GT) activity and catalyzes glycan chain elongation, whereas in class B, the N-terminal module is devoid of any known catalytic activity and is presumably involved in interactions with other proteins during cell morphogenesis (14). Membrane-bound monofunctional glycosyltransferases (MGTs) capable of catalyzing un-cross-linked glycan chain formation have been found in some bacteria (Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, Brucella abortus, Micrococcus luteus, Neisseria gonorrhoeae, and Ralstonia eutropha), but their exact role is less well understood. The GT domain of class A PBPs and the monofunctional GTs show high sequence similarity and belong to family GT51 in the sequence-based classification of GTs (7). They all possess five conserved motifs. Some MGTs have been produced, purified, and partially characterized. E. coli and S. aureus MGTs used lipid II (undecaprenyl-pyrophosphate-GlcNAc-MurNAc-
* Corresponding author. Mailing address: Centre d’Inge´nierie des Prote´ines, Universite´ de Lie`ge, Institut de Chimie, B6a, B-4000 SartTilman, Belgium. Phone: 32 4 3663397. Fax: 32 4 3663364. E-mail:
[email protected]. 2528
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of PG polymerization (2, 6). Owing to its location outside of the cytoplasmic membrane and its specificity, the glycosyltransferase is an interesting drug target that has not been fully explored yet. The development of new inhibitors requires the characterization of these enzymes at structural and mechanistic levels. Various constructions have been made to isolate the GT module of class A PBP1b. In each case, the activity of the GT module was ⬃20% of that of the full-size PBP (4, 21). MGTs appear to be of great interest for GT structure determination because of their smaller size compared to class A PBPs. However, no kinetic data are available on any MGT. The objective of this work was to determine the kinetic parameters of the S. aureus MGT and analyze the role of the putative catalytic Glu100 (equivalent to the catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The enzymatic properties of S. aureus MGT were compared to those of S. aureus PBP2, E. coli PBP1b, and E. coli MGT. MATERIALS AND METHODS Materials. The plasmid pNJ2⍀mgtSa, carrying S. aureus mgt gene (UniProt Q93Q23), was a gift from M. Arthur (1). The lipid II, undecaprenyl-pyrophosphoryl-MurNAc-(L-Ala-␥-D-Glu-meso-[14C]A2pm-D-Ala-D-Ala)-GlcNAc (A2pm is diaminopimelic acid), was prepared as described previously (23). Moenomycin was a gift from Aventis (Romainville, France). Construction of plasmids. (i) pDML2004. The truncated mgt gene encoding the (D68-R268) MGT was amplified by PCR using plasmid pNJ2⍀mgtSa as template (1) and the primers 5⬘-AATGCTGGTCATATGGATAATGTGGAT GAACTAAGAAAAATTG-3⬘ and 5⬘-CACCTCGAGGAATTCTTAACGATT TAATTGTGACATAGCCTG-3⬘. NdeI and EcoRI sites are underlined. The PCR fragment was digested by NdeI and EcoRI and inserted between the corresponding sites of pET28a(⫹) (Novagen, Madison, Wis.), giving rise to pDML2004. This construct resulted in the fusion of an N-terminal His tag to (D68-R268) MGT. The recombinant protein was called SauH6-MGT. (ii) pDML2004-E100Q. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene), the template plasmid pDML2004, and the primers 5⬘-CCTTTATTTCAATGCAAGATGAACGATT CTAC-3⬘ and 5⬘-GTAGAATCGTTCATCTTGCATTGAAATAAAGG-3⬘. The modified codon is underlined. The resulting plasmid was named pDML2004E100Q, and the protein was designated SauH6-MGT E100Q. Expression and purification of the SauH6-MGT and E100Q mutant. E. coli C41 (DE3) (15) was transformed with the plasmid pDML2004 or pDML2004E100Q, and the transformants were grown at 37°C in Luria-Bertani medium containing 50 g of kanamycin/ml. When the absorbance reached a value of 0.6 at 600 nm, the cultures were supplemented with 1 mM IPTG (isopropyl--Dthiogalactopyranoside) and grown for three additional hours. The cells were suspended in 50 mM sodium phosphate buffer (pH 7.0) containing 0.3 M NaCl (buffer P) and 1 mM PMSF (phenylmethylsulfonyl fluoride) and disrupted by sonication. After centrifugation at 40,000 ⫻ g at 4°C, the soluble fraction was loaded onto a 5-ml HisTrap Ni-Sepharose HP column (Amersham Biosciences) equilibrated with buffer P. The column was washed with buffer P containing, consecutively, 1 M NaCl (10 volumes), 40 mM imidazole and 0.3% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} (10 volumes), and finally 150 mM imidazole (10 volumes). The elution was performed in buffer P with an imidazole gradient from 150 to 300 mM. The protein (25 kDa) was dialyzed against 25 mM Tris-HCl (pH 7.5) containing 0.3 M NaCl and 10% glycerol (buffer G) and then concentrated to 2 mg/ml and stored at 4°C in the presence of 0.02% sodium azide. Glycosyltransferase reaction (glycan chain synthesis). Typical assays were performed by incubating meso-[14C]A2pm-labeled C55 lipid II (2.24 M; 0.126 Ci/nmole) and SauH6-MGT (64 nM) for 20 to 30 min at 30°C in 50 mM Tris-HCl, pH 7.5, supplemented with 10 mM MnCl2, 0.5% decyl polyethylene glycol (PEG), 12.5% 1-octanol, and 12.5% dimethyl sulfoxide (DMSO). The polymerized material was digested in the presence of 0.2 mg/ml lysozyme at 30°C for 30 min. The reaction products were separated by overnight chromatography on Whatman no. 1 filter paper in isobutyric acid–1 M ammonia (5:3) (12). Under these conditions, the polymerized peptidoglycan remained immobile on the chromatograms while the lipid II substrate and lysozyme digests migrated with Rf
FIG. 1. Expression and purification of SauH6-MGT. Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from C41 (DE3) cells producing SauH6MGT in the absence of IPTG (1) or in the presence of IPTG (2) and purified SauH6-MGT (2 g) (3). MS, molecular mass standard.
values of 0.9 and 0.3, respectively. The radioactive compounds were detected with a PhosphorImager scanner and analyzed with Quantity One software (Bio-Rad).
RESULTS Protein preparation and GT assay optimization. A soluble form of S. aureus MGT devoid of its membrane anchor, called SauH6-MGT (D68-R268), was expressed in the cytoplasm of E. coli C41 (DE3) strain (15) and purified on a HisTrap NiSepharose HP column (see Materials and Methods) (Fig. 1). The protein was 85% pure, and the final yield was about 5 mg/liter of culture. Gel filtration analysis in the presence of 25 mM Tris-HCl (pH 7.5)–0.3 M NaCl–10% glycerol (buffer G) showed that the protein was eluted with an apparent molecular mass of ⬃400 kDa, indicating an aggregation of the protein as described previously (24). When gel filtration was carried out in buffer G containing 0.7% CHAPS, most of the protein (80%) was eluted with the expected molecular mass of 25 kDa (data not shown). SauH6-MGT (D68-R268) catalyzed glycan chain polymerization when the protein (64 nM) was incubated in the presence of meso-[14C]A2pm-labeled C55 lipid II (2.24 M) in 50 mM Tris-HCl (pH 7.5) buffer containing 10 mM MnCl2, 0.5% decyl PEG, 12.5% 1-octanol, and 12.5% DMSO. We verified that the polymerized product was completely digested with lysozyme. Moenomycin, used at a concentration of 75 nM and an antibiotic/MGT ratio of 1.2:1, inhibited the GT activity by 50%. SauH6-MGT (D68-R268) was fivefold-less sensitive to moenomycin than E. coli PBP1b (75 nM versus 15 nM) (21). The optimal conditions for the activity of SauH6-MGT (D68-R268) were determined by using variable concentrations of 1-octanol, DMSO, or decyl PEG. Omitting one of these reagents in the reaction mixture resulted in very low GT activity. The optimal conditions are probably a compromise between C55-lipid II solubility and enzyme integrity in the pres-
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FIG. 2. Effects of Mn2⫹ and Mg2⫹ concentrations on the GT activity of SauH6-MGT (A) and pH dependence (B). The reaction was carried out at 30°C with SauH6-MGT (64 nM), 0.5% decyl PEG, 12.5% DMSO, 12.5% octanol, and 2.24 M meso-[14C]A2pm-labeled C55 lipid II. In addition, the assay mixtures contained 50 mM buffer and metal ions. The assay conditions were 50 mM Tris-HCl, pH 7.5, with variable concentrations of MnCl2 and MgCl2 (0.1 to 150 mM) (A) and 10 mM MnCl2 with different buffer conditions: sodium acetate (pH 5.0), piperazine-N,N⬘-bis(2-ethanesulfonic acid) or morpholineethanesulfonic acid (pH 6.0), HEPES (pH 7.0 to 8.0), and Tris-HCl (pH 7.5 to 8.5) (B).
ence of the mixture containing 1-octanol, DMSO, and decyl PEG detergent. Effect of the metal ions and pH on SauH6-MGT (D68-R268) activity. The effect of metal ions on the activity of SauH6-MGT was tested at pH 7.5. The results show that SauH6-MGT was almost inactive in the presence of EDTA or in the absence of metal ions (0.8 and 0.65 nmol of disaccharide U/min/mg, respectively). The GT activity of the enzyme was strongly stimulated in the presence of Mn2⫹ (18 nmol/min/mg), Ca2⫹ (13 nmol/min/mg), or Mg2⫹ (12 nmol/min/mg) at a concentration of 10 mM. Effects of various concentrations of Mn2⫹ and Mg2⫹ on the enzyme activity were also determined (Fig. 2A). The results show that the concentrations of Mn2⫹ or Mg2⫹ needed for optimal SauH6-MGT activity were 10 mM and 50 mM, respectively, indicating that Mn2⫹ was a better cofactor for the enzyme activity than Mg2⫹. The pH dependence study was carried out at various pHs ranging from 5 to 8.5 in the presence of 10 mM MnCl2. Figure 2B shows that the activity of SauH6-MGT was optimal at pH 7.5. Kinetic parameters of SauH6-MGT (D68-R268). To determine the kinetic parameters of SauH6-MGT (D68-R268), the enzyme (64 nM) was incubated in the presence of meso[14C]A2pm-lipid II substrate at concentrations varying from 0.25 to 5 M, at pH 6.0 with Mn2⫹ and at pH 7.5 with Mn2⫹
or Mg2⫹. Analysis of the initial rate measurements of radioactive peptidoglycan synthesis and application of the MichaelisMenten equation permitted determination of the kinetic parameters of the reaction (Table 1). At pH 7.5 and in the presence of Mg2⫹ (10 mM or 50 mM), the Km values were 1.9 ⫾ 0.8 and 1.5 ⫾ 0.5 M and the kcat values were (8 ⫾ 2.0) ⫻ 10⫺3 s⫺1 and (7.6 ⫾ 1.0) ⫻ 10⫺3 s⫺1, respectively. Therefore, the kcat/Km efficiencies were ⬃4,400 M⫺1 s⫺1 at 10 mM and 5,100 M⫺1 s⫺1 at 50 mM of Mg2⫹. At pH 7.5 and in the presence of Mn2⫹ (10 mM), the reaction proceeded with a Km value of 2.2 ⫾ 1.4 M, a kcat value of (13 ⫾ 4) ⴛ 10⫺3 s⫺1, and a kcat/Km of ⬃5,800 M⫺1 s⫺1. At pH 6.0 and in the presence of Mn2⫹, the Km value was 2.8 ⫾ 2.0 M, the kcat value was (6.3 ⫾ 2.6) ⫻ 10⫺3 s⫺1, and the kcat/Km efficiency was ⬃2,100 M⫺1 s⫺1. This result shows that the activity of the enzyme is higher at pH 7.5 than at pH 6.0. The effect of pH 7.5 versus pH 6.0 seems to affect mainly the kcat value, which increased about twofold. Role of the conserved Glu100 amino acid residue. The Glu residue in motif 1 is strictly conserved in all class A PBPs and MGTs. We have previously shown that Glu233 is essential for the GT activity of E. coli PBP1b (21). In order to test the function of Glu100 in motif 1 of S. aureus MGT, this residue was replaced by Gln. The SauH6-MGT Glu100Gln mutant was
TABLE 1. Kinetic parameters of SauH6-MGT with lipid II substratea S. aureus MGT Parameter
kcat (10⫺3 s⫺1) Km (M) kcat/Km (M⫺1 s⫺1)
10 mM Mn (pH 6.0)
2⫹
6.3 ⫾ 2.6 2.8 ⫾ 2.0 2,100
10 mM Mn2⫹ (pH 7.5)
10 mM Mg2⫹ (pH 7.5)
50 mM Mg2⫹ (pH 7.5)
E. coli PBP1bb
S. aureus PBP2c
13.0 ⫾ 4.0 2.2 ⫾ 1.4 5,800
8.0 ⫾ 2.0 1.9 ⫾ 0.8 4,400
7.6 ⫾ 1.0 1.5 ⫾ 0.5 5,100
70 ⫾ 13 1.8 ⫾ 0.8 39,000
15.0 ⫾ 1.0 4.0 ⫾ 1.0 3,400
a The reactions were carried out in 50 mM PIPES (pH 6.0) or 50 mM Tris-HCl (pH 7.5) containing 10 or 50 mM metal ion, 0.5% decyl PEG, 12.5% DMSO, 12.5% l-octanol, 64 nM SauH6-MGT, and 0.25 to 5 M meso-[14C]A2pm-labeled C55 lipid II. b Data are from reference 21. c Data are from reference 3.
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expressed and purified as described for the wild-type SauH6MGT without a detectable difference in the behavior of the two proteins in terms of stability, except that the expression level of the mutant was four times higher than that of the wild type, presumably because the mutant has a less deleterious effect on bacterial growth than the nonmutated protein. The glycosyltransferase activity of the SauH6-MGT Glu100Gln mutant was reduced 500-fold compared to the wild type (23.3 nmol of lipid II used/mg of enzyme/min versus 11.6 ⫻ 103 nmol/mg/min). This result is similar to that obtained with the E. coli PBP1b E233Q mutant, which in addition was not active in vivo (21). Therefore, although the Glu100Gln mutant retains very low activity, the Glu100 residue may also be essential for the physiological activity of S. aureus MGT in vivo. DISCUSSION A very limited number of GTs have been purified and characterized (E. coli PBP1b, S. aureus PBP2, and S. pneumoniae PBP2a) and among them no MGTs. To our knowledge this is the first detailed characterization (kinetic parameters, metal ion, and pH effects) of an MGT using purified enzyme and purified lipid II substrate. We produced a soluble form of the S. aureus MGT devoid of its membrane anchor similar to that previously reported (24); both constructs contain the polypeptide D68-R268 but differ slightly in the added N-terminal polyHis-containing segments (H6 and H10, respectively). H10-MGT was expressed in E. coli BL21(DE3)/pLysS, whereas SauH6MGT could not be overexpressed in this strain but was highly expressed in E. coli C41 BL21(DE3) (15). As determined in the in vitro GT assay developed for E. coli PBP1b and purified meso-[14C]A2pm-labeled lipid II as substrate, SauH6-MGT was able to catalyze glycan chain polymerization. This precursor contains A2pm in the third position of the peptide moiety instead of penta-Gly-substituted Lys, the natural substrate in S. aureus. These results show that the integrity of the peptide sequence is not an absolute requirement for the GT activity, but we cannot rule out the possibility that the peptide may have a role in interaction with the enzyme. These questions can be answered by using lipid II containing different peptide moieties. In contrast to E. coli MGT, which was insensitive to moenomycin (8), the GT activity catalyzed by SauH6-MGT was inhibited by this inhibitor, as previously shown (24). The inhibition efficiency was about fivefold lower than for E. coli PBP1b (75 nM versus 15 nM) (21). It is possible that moenomycin makes interactions beyond the GT domain of PBP1b (the additional N-terminal insertion or transpeptidase domain), increasing its affinity for this protein. In a previous study, the enzymatic activity of the purified S. aureus H10-MGT was measured on the basis of the incorporation of 14C-labeled N-acetylglucosamine using the membrane fraction of Aerococcus viridans (24). On the other hand, the enzymatic activity of E. coli MGT was measured directly with 14 C-labeled lipid II substrate but the enzyme was not purified (8). S. aureus H10-MGT and E. coli MGT have optimal activity at pH 6.0. Compared to our GT reaction, where both lipid II and the SauH6-MGT enzyme were purified and the GT assay was optimized, the previous studies used a complex mixture; this may explain the differences in the enzyme activities in pH function in the two studies (see below). S. aureus PBP2 also
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had a low pH optimum of 5.0 (3). Furthermore, S. aureus PBP2 was active in the absence of metal ions, and the addition of ions had a moderate effect on activity (3). In contrast, we found that SauH6-MGT had a pH optimum of 7.5 and that the protein was inactive in the absence of metal ions; addition of Mg2⫹, Mn2⫹, or Ca2⫹ drastically enhanced the activity of the enzyme. The SauH6-MGT properties determined here are similar to those of E. coli PBP1b in terms of pH optimum and metal ion requirement, although PBP1b has a preference for Ca2⫹ (20) whereas SauH6-MGT seems to prefer Mn2⫹. The enzymatic efficiency of SauH6-MGT, the kcat/Km value, is about 5,800 M⫺1 s⫺1 (with a Km value of 2.2 M and a kcat value of 13 ⫻ 10⫺3 s⫺1). The catalytic efficiency is 7.5-fold lower than that of E. coli PBP1b and similar to that of S. aureus PBP2 (Table 1). The kcat value of S. aureus MGT was close to that of S. aureus PBP2 (13 ⫻ 10⫺3 versus 15 ⫻ 10⫺3 s⫺1) but fivefold lower than that of E. coli PBP1b (13 ⫻ 10⫺3 versus 70 ⫻ 10⫺3 s⫺1). The catalytic constant kcat with Mn2⫹ was 1.6-fold that with Mg2⫹, suggesting that the metal ion may play a catalytic role in the reaction catalyzed by SauH6-MGT, as has been shown for E. coli PBP1b (20). The Km value of SauH6MGT was comparable to those of PBP1b (2.2 versus 1.8 M) and S. aureus PBP2 (2.2 versus 4 M). This observation may suggest that the contribution of the peptide moiety of lipid II in overall binding between the substrate and the bifunctional enzymes is probably weak. The glutamate residue of the first conserved motif, EDXXFXX(H/N)X(G/A), is highly conserved in all known GT51 sequences (P. M. Coutinho and B. Henrissat, Carbohydrate-Active Enzymes server [http://afmb.cnrs-mrs.fr/CAZY /GT_51.html]). In E. coli PBP1b, E233 in this position was found by site-directed mutagenesis to be essential for catalysis, and a model based on the glycosidase mechanism was proposed (21). Further investigations of PBP1b GT activity dependence on pH and metal ions allowed the proposition of a model wherein Glu233 catalyzes deprotonation of the 4-OH nucleophile of the growing glycan chain while a metal ion stabilizes the leaving group (20). In the present study, we found that S. aureus MGT presents a pH optimum and metal ion requirement similar to that of E. coli PBP1b. In addition, the conserved Glu100 was important for the activity of S. aureus MGT, confirming the essential role of the glutamate residue of the first motif, EDXXFXX(H/N)X(G/A), and suggesting that it is likely the key catalytic residue of the GT51 active site. For more than two decades, E. coli PBP1b, a multimodular class A PBP, has been the source of knowledge on the GT reaction (16, 20, 21, 23). While this protein is a useful tool for biochemical studies, its properties, which are probably common to many other class A PBPs (10), have until now prevented the growth of useful crystal and determination of its structure. The SauH6-MGT characterized in this work displays relatively high GT activity, indicating that the native GT domain is properly folded. Its properties, in terms of solubility and small size, could make it an appropriate candidate for structural and mechanistic studies. ACKNOWLEDGMENTS This work was supported by Return Grant to M.T. from the Belgian Science Policy and was partially supported by the Belgian Program on Interuniversity Poles of Attraction, initiated by the Belgian State,
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` CHE TERRAK AND NGUYEN-DISTE
Prime Minister’s Office, Science Policy Programming (IAP P5/33); the Actions de Recherche Concerte´es (grant 03/08-297); and the European Commission (grant LSMH-CT-2003-503335). We thank J.-M. Fre`re for reading the manuscript. REFERENCES 1. Arbeloa, A., H. Segal, J. E. Hugonnet, N. Josseaume, L. Dubost, J. P. Brouard, L. Gutmann, D. Mengin-Lecreulx, and M. Arthur. 2004. Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis. J. Bacteriol. 186:1221–1228. 2. Baizman, E. R., A. A. Branstrom, C. B. Longley, N. Allanson, M. J. Sofia, D. Gange, and R. C. Goldman. 2000. Antibacterial activity of synthetic analogues based on the disaccharide structure of moenomycin, an inhibitor of bacterial transglycosylase. Microbiology 146:3129–3140. 3. Barrett, D., C. Leimkuhler, L. Chen, D. Walker, D. Kahne, and S. Walker. 2005. Kinetic characterization of the glycosyltransferase module of Staphylococcus aureus PBP2. J. Bacteriol. 187:2215–2217. 4. Barrett, D. S., L. Chen, N. K. Litterman, and S. Walker. 2004. Expression and characterization of the isolated glycosyltransferase module of Escherichia coli PBP1b. Biochemistry 43:12375–12381. 5. Canavessi, A. M., J. Harms, N. de Leon Gatti, and G. A. Splitter. 2004. The role of integrase/recombinase XerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo. Microb. Pathog. 37:241–251. 6. Chen, L., D. Walker, B. Sun, Y. Hu, S. Walker, and D. Kahne. 2003. Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate. Proc. Natl. Acad. Sci. USA 100: 5658–5663. 7. Coutinho, P. M., E. Deleury, G. J. Davies, and B. Henrissat. 2003. An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328:307–317. 8. Di Berardino, M., A. Dijkstra, D. Stuber, W. Keck, and M. Gubler. 1996. The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan-synthesising enzymes. FEBS Lett. 392:184–188. 9. Di Guilmi, A. M., A. Dessen, O. Dideberg, and T. Vernet. 2003. The glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae catalyzes the polymerization of murein glycan chains. J. Bacteriol. 185:4418–4423. 10. Di Guilmi, A. M., N. Mouz, L. Martin, J. Hoskins, S. R. Jaskunas, O. Dideberg, and T. Vernet. 1999. Glycosyltransferase domain of penicillinbinding protein 2a from Streptococcus pneumoniae is membrane associated. J. Bacteriol. 181:2773–2781. 11. Goffin, C., and J. M. Ghuysen. 1998. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol. Rev. 62:1079–1093.
J. BACTERIOL. 12. Hara, H., and H. Suzuki. 1984. A novel glycan polymerase that synthesizes uncross-linked peptidoglycan in Escherichia coli. FEBS Lett. 168:155–160. 13. Harper, M., J. D. Boyce, I. W. Wilkie, and B. Adler. 2003. Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens. Infect. Immun. 71:5440– 5446. 14. Marrec-Fairley, M., A. Piette, X. Gallet, R. Brasseur, H. Hara, C. Fraipont, J. M. Ghuysen, and M. Nguyen-Disteche. 2000. Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli. Mol. Microbiol. 37:1019–1031. 15. Miroux, B., and J. E. Walker. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260:289–298. 16. Nakagawa, J., S. Tamaki, S. Tomioka, and M. Matsuhashi. 1984. Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides. Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase. J. Biol. Chem. 259:13937–13946. 17. Palavecino, E. 2004. Community-acquired methicillin-resistant Staphylococcus aureus infections. Clin. Lab. Med. 24:403–418. 18. Pinho, M. G., S. R. Filipe, H. de Lencastre, and A. Tomasz. 2001. Complementation of the essential peptidoglycan transpeptidase function of penicillin-binding protein 2 (PBP2) by the drug resistance protein PBP2A in Staphylococcus aureus. J. Bacteriol. 183:6525–6531. 19. Schiffer, G., and J. V. Holtje. 1999. Cloning and characterization of PBP 1C, a third member of the multimodular class A penicillin-binding proteins of Escherichia coli. J. Biol. Chem. 274:32031–32039. 20. Schwartz, B., J. A. Markwalder, S. P. Seitz, Y. Wang, and R. L. Stein. 2002. A kinetic characterization of the glycosyltransferase activity of Escherichia coli PBP1b and development of a continuous fluorescence assay. Biochemistry 41:12552–12561. 21. Terrak, M., T. K. Ghosh, J. van Heijenoort, J. Van Beeumen, M. Lampilas, J. Aszodi, J. A. Ayala, J. M. Ghuysen, and M. Nguyen-Disteche. 1999. The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli. Mol. Microbiol. 34:350–364. 22. van Heijenoort, J. 2001. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology 11:25R–36R. 23. van Heijenoort, Y., M. Gomez, M. Derrien, J. Ayala, and J. van Heijenoort. 1992. Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3. J. Bacteriol. 174:3549–3557. 24. Wang, Q. M., R. B. Peery, R. B. Johnson, W. E. Alborn, W. K. Yeh, and P. L. Skatrud. 2001. Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus. J. Bacteriol. 183:4779–4785.
