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Journal of Bacteriology, November 2008, p. 7141-7146, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00676-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of the Bacterial UDP-GlcNAc:Undecaprenyl-Phosphate GlcNAc-1-Phosphate Transferase WecA

Bayan Al-Dabbagh,¹ Dominique Mengin-Lecreulx,^1,2 and Ahmed Bouhss^1,2*

Université Paris-Sud, UMR 8619, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Orsay, France,¹ CNRS, UMR 8619, Laboratoire des Enveloppes Bactériennes et Antibiotiques, Orsay, France²

Received 14 May 2008/ Accepted 2 August 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The first membrane step of the biosynthesis of various polymers of the bacterial cell wall such as the O-antigen and enterobacterial common antigen is catalyzed by the UDP-N-acetylglucosamine (UDP-GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA (21, 23, 34, 35). It consists in the transfer of the phospho-GlcNAc moiety from the nucleotide precursor onto a lipid carrier, undecaprenyl-phosphate (C₅₅-P), leading to the formation of C₅₅-PP-GlcNAc (lipid intermediate I) with the subsequent release of UMP (22, 31). This reaction absolutely requires a divalent metal ion, such as Mg²⁺ or Mn²⁺ (3).

The WecA enzyme is an integral membrane protein (4) whose membrane topology has been recently determined (18). It is composed of 11 transmembrane segments, five cytoplasmic domains, and five periplasmic domains. The N- and C-terminal ends of this protein have been shown to be located in the periplasm and the cytoplasm, respectively, based on evidence obtained with PhoA and LacZ fusions (18). Sequence analysis of bacterial WecA proteins shows that the five cytoplasmic domains contain many conserved amino acid residues including several invariant aspartate and histidine residues important for enzymatic activity (18). The three central cytoplasmic sequences (II, III, and IV) of WecA were also found in other prokaryotic and eukaryotic proteins catalyzing the transfer of a lipid phosphate carrier to the β-phosphate of a UDP-linked hexosamine. These proteins belong to a superfamily of enzymes called polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferases (8). This superfamily includes bacterial members such as MraY, TagO, WbcO, WbpL, and RpgG implicated in the biosynthesis of different cell envelope polymers (peptidoglycan, teichoic acids, or rhamnose-glucose polysaccharide) (6, 29, 30, 33, 36) as well as the eukaryotic UDP-GlcNAc:dolichyl-P GlcNAc-1-P transferase (GPT) that catalyzes the transfer of phospho-GlcNAc to dolichyl phosphate initiating the N-linked glycoprotein biosynthesis (9, 13, 19). Bacterial members of this superfamily share a common membrane-bound acceptor substrate, C₅₅-P. However, they utilize different UDP-N-acetyl-hexosamine substrates and differ by their susceptibilities to inhibitors targeting this enzyme family (5, 29).

WecA orthologues from different bacterial species are generally composed of 350 to 360 amino acid residues. Larger WecA proteins having an extension of ca. 200 residues at the C terminus are found in Pirellula baltica and Nitrosomonas europaea (532 and 540 amino acid residues, respectively). Smaller WecA proteins are also encountered in the thermophilic bacteria Thermus thermophilus and Thermotoga maritima (323 and 291 residues, respectively). Despite this variability in the sequence length of these protein orthologues, their global membrane topologies are predicted to be similar to the topology experimentally determined for Escherichia coli WecA (18).

To date, all reported studies on WecA involved only crude membrane preparations as the source of this bacterial enzyme (2, 3, 18, 31). In the present work and for the first time, a WecA enzyme was overproduced, extracted from membranes with a detergent, and purified to near homogeneity, and its biochemical properties were investigated in detail.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains, plasmids, and growth conditions. The E. coli strains DH5 (Invitrogen) and C43(DE3) (Avidis) were used as hosts for plasmids as well as for the overproduction of the WecA enzyme. 2YT medium (24) was used as a culture medium, and growth was monitored at 600 nm with a Shimadzu UV-1601 spectrophotometer. Ampicillin was used at a concentration of 100 µg · ml^–1.

Chemicals. DNA restriction and modification enzymes were obtained from New England Biolabs, and oligonucleotides were from MWG-Biotech. Farnesyl phosphate (C₁₅-P); geranylgeranyl phosphate (C₂₀-P), hexaprenyl phosphate (C₃₀-P), heptaprenyl phosphate (C₃₅-P), octaprenyl phosphate (C₄₀-P), C₅₅-P, dolichyl(C₅₅)-P (an analogue of C₅₅-P with a saturated bond between C₂ and C₃), dodecaprenyl phosphate (C₆₀-P), pentadecaprenyl phosphate (C₇₅-P), and undecaprenol (C₅₅-OH) were provided by the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warszaw, Poland. Ni²⁺-nitrilotriacetic acid (Ni²⁺-NTA) agarose was from Qiagen and isopropyl-β-D-thiogalactopyranoside (IPTG) was from Eurogentec. n-Dodecyl-β-D-maltoside (DDM) was from Anatrace; Tween 20 was from VWR; and n-decyl-β-D-maltoside, Triton X-100, n-octyl-β-D-glucopyranoside, tunicamycin, and UDP-GlcNAc were from Sigma. UDP-[¹⁴C]GlcNAc was purchased from Perkin-Elmer Life Sciences, and UDP-N-acetyl-muramyl-L-Ala--D-Glu-meso-diaminopimeloyl-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and lipid intermediate II of peptidoglycan were purified as described previously (6). All other materials were reagent grade and obtained from commercial sources.

Plasmid constructions. Standard procedures for molecular cloning (32) and cell transformation (12) were used. The T. maritima wecA gene was amplified by PCR from the chromosome of strain MSB8; for this purpose, primers 5'-AGGCACGGATCCATGTGGGAAGCGATAATTAGTTTCTTCC-3' and 5'-ATACCAAAGCTTTTACAGCTTGAGGTTGCCATTACC-3' containing a BamHI and a HindIII site (in bold type), respectively, were employed. The PCR fragment was purified using a Wizard PCR Preps DNA purification kit (Promega); the fragment was then digested by BamHI and HindIII and inserted between the same sites of the pET2130 plasmid vector (T7 promoter) (10), generating the plasmid pWTM8. In this construct, the wecA gene from T. maritima was expressed under the control of a strong IPTG-inducible promoter, and the encoded WecA protein carried a Met-His₆-Gly-Ser N-terminal extension. DNA sequencing was performed to ensure that the sequence of the cloned fragment was correct (MWG-Biotech).

Preparation of crude enzyme. E. coli C43(DE3) cells harboring the recombinant plasmid pWTM8 were grown at 37°C in 2YT-ampicillin medium (2-liter culture). At an A₆₀₀ of 0.7, IPTG was added at a final concentration of 1 mM, and incubation was continued for 16 h at 25°C with shaking. Cells were harvested by centrifugation (8,000 x g for 20 min at 4°C), washed in 100 ml of 25 mM Tris-HCl buffer, pH 7.5, and resuspended in 5 ml of the same buffer containing 2 mM 2-mercaptoethanol, 150 mM NaCl, and 10% glycerol (buffer A). Cells were disrupted by sonication in the cold (Bioblock Vibracell sonicator, model 72412), and the resulting suspension was centrifuged at 10°C for 30 min at 200,000 x g in a Beckman TL100 centrifuge. The pellet consisting of membranes and associated proteins (4.1 g wet weight; 408.5 mg of proteins) was washed three times with buffer A and then subjected to solubilization by detergents as described below.

Solubilization of WecA. Membrane vesicles containing the overexpressed WecA protein were resuspended in 10 ml of buffer A. DDM was added at a final concentration of 49 mM, and the mixture was incubated at 4°C for 2 h with shaking. After centrifugation at 200,000 x g for 30 min at 4°C, the supernatant was recovered. The same procedure was used for extraction with other detergents, and the final concentration of detergent was 137 and 124 mM for n-octyl-β-D-glucopyranoside and Triton X-100, respectively.

Purification of histidine-tagged WecA. Solubilized membrane proteins were mixed and incubated for 2 h at 4°C with Ni²⁺-NTA agarose (Qiagen) (15 mg of protein/ml of resin) preequilibrated in 25 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl, 10% glycerol, 3.9 mM DDM, and 2 mM 2-mercaptoethanol (buffer B). After incubation, the resin was transferred to an Econo-Pac chromatography column (Bio-Rad). Washings and protein elution were performed with increasing concentrations of imidazole, from 7.5 to 200 mM, in buffer B. Elution of the protein was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic assays. Pure protein-containing fractions were freed from imidazole by dialysis against 30 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 10% glycerol, and 3.9 mM DDM. The same procedure was used for purification of WecA with the detergents n-octyl-β-D-glucopyranoside and Triton X-100 (used at 68.5 and 15.5 mM, respectively).

Protein monitoring. Protein concentrations were determined by using a QuantiPro bicinchoninic acid assay kit (Sigma) with bovine serum albumin as the standard.

Assays for transferase activity. (i) Standard WecA assay. A standard WecA assay was performed in a reaction mixture (10 µl) containing, in final concentrations, 100 mM Tris-HCl buffer, pH 8, 10 mM MgCl₂, 1.1 mM C₅₅-P, 0.16 mM UDP-[¹⁴C]GlcNAc (550 Bq), and 92.7 mM Triton X-100. The reaction was initiated by the addition of WecA enzyme (ca. 5 ng), and the mixture was incubated for 30 min at 65°C. For the determination of the K_m values, the WecA activity was assayed as described above with various concentrations of one substrate (0.16 mM to 3 mM for UDP-GlcNAc; 0.05 mM to 1.1 mM for C₅₅-P), while maintaining the other at a fixed value (1.1 mM for C₅₅-P; 0.16 mM for UDP-GlcNAc). Data were fitted to the equation v = VA/(K + A) (where v is the experimentally determined rate, V is the maximum velocity, A is the substrate concentration, and K is the Michaelis constant) by using the MDFitt software developed by M. Desmadril (UMR 8619 CNRS, Orsay, France). Results are expressed as mean ± standard deviation of three independent experiments. Other potential lipid substrates of WecA were also tested: C₁₅-P, C₂₀-P, C₃₀-P, C₃₅-P, C₄₀-P, C₆₀-P, and C₇₅-P, under the same standard conditions described above.

(ii) MraY assay. The standard MraY assay (6) was carried out in a volume of 10 µl containing 100 mM Tris-HCl, pH 8, 10 mM MgCl₂, 1.1 mM C₅₅-P, and 0.25 mM UDP-MurNAc-[¹⁴C]pentapeptide (337 Bq). The reaction was initiated by the addition of the protein (ca. 5 ng), and the mixture was incubated for 30 min at 37°C or 65°C.

In all cases, the reaction was stopped by heating at 100°C for 1 min, and the radiolabeled substrate and product, UDP-GlcNAc and C₅₅-PP-GlcNAc or UDP-MurNAc-pentapeptide and C₅₅-PP-MurNAc-pentapeptide for WecA and MraY proteins, respectively, were separated by thin-layer chromatography on silica gel plates LK6D (Whatman) using 2-propanol-ammonium hydroxide-water (6:3:1; vol/vol/vol) as a mobile phase. The radioactive spots were located and quantified with a radioactivity scanner (model Multi-Tracemaster LB285; Berthold-France). One unit of enzyme activity corresponds to one nanomole of lipid I formed per min, and specific activities are expressed as units per milligram of protein.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The wecA gene has been demonstrated to catalyze the first membrane step of O-antigen and enterobacterial common antigen biosyntheses in E. coli (1). Its involvement in the virulence of gram-negative bacteria has been reported (16, 26, 27). Thus, the WecA transferase can be considered as a promising potential target to be exploited for new antibacterial discovery. To date, this enzyme has been clearly underexploited, most probably because of the refractory nature of this protein to overexpression and purification. All previous attempts to overexpress significantly and purify any WecA protein had been unsuccessful. Therefore, only crude membrane preparations were generally used for enzymatic assays (18).

We undertook the overproduction and purification of WecA from T. maritima in order to take this protein from this gram-negative, extremely thermophilic bacterium as a model for biochemical and structural characterization of this family of enzymes.

Overproduction and purification of WecA from T. maritima. The T. maritima wecA gene was amplified from the chromosome of strain MSB8 and cloned into pET2130, generating the plasmid pWTM8. The sequence of the cloned gene was verified and shown to be identical to that found in databases. In this construct, the wecA gene product was expressed with an N-terminal polyhistidine tag to allow its easy purification on Ni²⁺-NTA agarose. Taking into account the N-terminal extension consisting in Met-His₆-Gly-Ser, the molecular mass calculated for the T. maritima WecA protein was 33,876 Da. Strain C43(DE3), which is particularly well adapted for high-level expression of membrane proteins (25), was chosen as the host strain. Cells of C43(DE3)(pWTM8) were grown at 37°C in 2YT-ampicillin medium and then induced at 25°C with 1 mM IPTG.

Small-scale cultures (50 ml) were performed, and membrane proteins of IPTG-induced cells were solubilized and tested for WecA activity. Three different detergents (DDM, Triton X-100, and n-octyl-β-D-glucopyranoside) were tested for their efficiency to extract the WecA protein from the membranes. The supernatants recovered after high-speed centrifugation of detergent extracts were tested for WecA activity. The recovered WecA activity was 96, 38, and 69% for DDM, Triton X-100 and n-octyl-β-D-glucopyranoside, respectively, compared to the total enzymatic activity in crude membranes (4.5 units/mg of protein). Clearly, DDM appeared more efficient than the other detergents for WecA extraction. At this step, however, no significant increase of a protein band that could correspond to WecA (calculated mass of about 34 kDa) was detected by SDS-PAGE analysis of the extracts. In contrast to membranes and detergent extracts prepared from bacteria harboring pWTM8, no WecA activity was detected in the membranes prepared from cells carrying the empty vector pET2130 (data not shown). Extracts containing the overexpressed WecA activity were further purified on Ni²⁺-NTA agarose in the presence of the same detergent that had been used for the protein extraction. Washing and elution steps were carried out with a discontinuous gradient of imidazole (from 7.5 to 200 mM), and fractions were analyzed for protein content and WecA transferase activity. The purification states (as judged by SDS-PAGE) were quite similar for the three tested detergents (data not shown). However, the specific WecA activity appeared 12- and 20-fold higher with DDM (261 units) than with Triton X-100 (21.8 units) and n-octyl-β-D-glucopyranoside (13.2 units), respectively. DDM thus appeared as the most efficient detergent for both the extraction and purification steps of the WecA protein. Therefore, it was chosen for a large-scale purification of the T. maritima WecA protein and enzyme characterization experiments.

A 2-liter culture of E. coli C43(DE3)(pWTM8) was performed, and the WecA protein was extracted from cell membranes by the detergent DDM and purified by affinity chromatography as described above. The WecA activity started to be released from the Ni²⁺-NTA resin at 30 mM imidazole, but the specific activity was shown to be maximal in the 200 mM imidazole-containing fraction (Table 1). Analysis of the latter purified fractions by SDS-PAGE showed a unique protein band migrating as a protein of 30 kDa (Fig. 1), a value slightly lower than the calculated one (33,876 Da). However, such a difference is often observed with integral membrane proteins (6, 7, 15, 28). This purification procedure yielded about 1.5 mg of pure WecA protein per liter of culture, with a specific activity of 290 units/mg of protein.

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TABLE 1. Purification of the T. maritima WecA protein from the DDM extract on Ni2+-NTA agarosea

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FIG. 1. SDS-PAGE of purified T. maritima WecA protein. The WecA protein was overproduced in E. coli cells in the His6-tagged form. The one-step purification on Ni2+-NTA agarose was performed as described in the text, and aliquots were analyzed by SDS-PAGE. Lane 2, DDM extract; lane 3, flowthrough; lane 4, 200 mM imidazole-containing fraction. Molecular mass standards indicated on the left (lane 1) are as follows: phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; and lysozyme, 14 kDa. The arrow points to the purified WecA protein. Staining was performed with Coomassie brilliant blue R250 (Merck).

Development of a WecA transferase assay. A new convenient WecA enzymatic assay was developed in this study. It is based on the addition of the phospho-[¹⁴C]GlcNAc moiety from UDP-[¹⁴C]GlcNAc onto C₅₅-P, followed by thin-layer chromatography separation on silica gel plates. Radioactive spots corresponding to the nucleotide substrate and lipid product (R_f of 0.39 and 0.81, respectively) were quantified with a radioactivity scanner. When C₅₅-P was omitted in the reaction mixture or replaced by C₅₅-OH, no WecA activity was observed, demonstrating the absence of C₅₅-P carrier lipid contamination in the preparation. Moreover, no MraY activity was detected in the purified WecA preparation, demonstrating the absence of any traces of this paralogue, which initiates the membrane steps of peptidoglycan biosynthesis (6, 8), in this preparation. When crude membrane extracts instead of pure WecA were tested at 37°C in the standard conditions, a small additional peak (R_f of 0.6) of radiolabeled compound was observed that corresponded to peptidoglycan lipid intermediate II (data not shown), the product of the reaction catalyzed by MurG (11). However, this peak was absent when the pure WecA protein was tested, confirming that our preparation of WecA was also not contaminated by the MurG enzyme or by the peptidoglycan lipid intermediate I.

Biochemical properties of the pure WecA enzyme. The transferase activity of the pure enzyme preparation was characterized in more detail. First, the effects of pH, salts, metal ions, detergent concentration, and temperature were tested. The effect of pH was determined in the range 6.0 to 9.4 by using two different buffers. As shown in Fig. 2A, a classical bell-shaped curve was obtained, and the activity was optimal around pH 8.0. All subsequent experiments were performed at this pH value. The effect of salts was investigated with NaCl and KCl in the range of 0 to 1 M. WecA behaved similarly with respect to these two salts, both of which had an inhibitory effect on the enzymatic activity (Fig. 2B). Metal ions such as Mg²⁺ and Mn²⁺ may be involved as cofactors in the phosphoryl transfer reaction for enzymes catalyzing the formation of phosphodiester bonds (20). The WecA activity showed an absolute requirement for the divalent cation (Mg²⁺) (Fig. 2C) (18). No activity at all could be detected in the absence of metal ion, and the activity was optimal with Mg²⁺ at a concentration of 10 mM. However, at high concentrations, Mg²⁺ had an inhibitory effect, and, particularly, no activity was detected at 250 mM.

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FIG. 2. Biochemical properties of the pure WecA enzyme. The effects of different parameters on WecA enzyme activity were investigated: pH (A); the salts NaCl () and KCl () (B); MgCl2 (C); the detergents Triton X-100 (), C12E6 (), n-octyl-β-D-glucopyranoside (), Tween 20 (), DDM (), N-lauroyl sarcosine () and n-decyl-β-D-maltoside (•) (D); and temperature (E). A standard assay was carried out as described in the text with the following modifications: in panel A, bis-Tris-HCl buffer was used for pH 6.0 to 7.0, and Tris-HCl buffer was used for pH 7.2 to 9.4. Activity values are expressed in nmol/min/mg of protein. Each data point represents the mean of three independent experiments, and the standard deviation was in all cases less than 10%.

The effects of various detergents on the WecA activity were also tested (Fig. 2D). Triton X-100 clearly appeared as the most efficient one, with an optimal concentration ranging between 50 and 120 mM. A similar activity of WecA was observed in the presence of the detergents C₁₂E₆ or n-octyl-β-D-glucopyranoside (optimal concentrations of 90 to 200 mM and 70 to 100 mM, respectively). In the presence of N-lauroyl sarcosine, Tween 20, or DDM (optimal concentrations of 17, 50, and 80 to 100 mM, respectively), the WecA activity represented about 12 to 17% of that observed in the presence of Triton X-100. Among the different detergents tested, n-decyl-β-D-maltopyranoside appeared to be the worst as the enzyme activity at the optimal concentration of this detergent (10 to 15 mM) was ca. 20-fold lower than that observed with Triton X-100 (Fig. 2D). The WecA activity detected was probably the result of multiple interactions between enzyme-detergent and C₅₅-P-detergent complexes within detergent micelles. Triton X-100 was chosen as the detergent for the standard WecA assay. The enzymatic activity of WecA was also tested at different temperatures (Fig. 2E) and was shown to be optimal at 65°C, which is close to the optimal growth temperature of T. maritima. The protein was totally inactive at 80°C, and its activity at 30°C represented only ca. 7% of the optimal value.

Using these optimal assay conditions for WecA activity, the determination of the kinetic constants was carried out. Typical Michaelis-Menten kinetics were observed for the two substrates in the concentration ranges considered (0.16 to 3 mM for UDP-GlcNAc and 0.05 to 1.1 mM for C₅₅-P). The K_m values of the purified WecA for UDP-GlcNAc and C₅₅-P were 0.62 ± 0.13 mM and 0.12 ± 0.03 mM, respectively, and the catalytic constant k_cat was 72.1 ± 10.8 min^–1.

Substrate specificity of the pure WecA enzyme. In order to determine the importance of the carbon chain length of the lipid substrate on the activity of the purified WecA from T. maritima, the enzyme was tested in the presence of polyisoprenyl phosphate of different sizes, from C₁₅-P to C₇₅-P. No activity was detected in the case of C₁₅-P and C₂₀-P. In the presence of C₃₀-P, an activity of 3% was observed compared to that in the presence of the natural lipid substrate C₅₅-P. However, the enzyme was highly active when tested with C₃₅-P, C₄₀-P, C₆₀-P, and C₇₅-P (Fig. 3). Thus, these results suggested that at least a 35-carbon chain was required for the lipid to be a substrate of WecA. Dolichyl(C₅₅)-P, a reduced analogue of C₅₅-P with a saturated bond between C₂ and C₃, was also efficiently used by WecA, with the activity observed representing about 60% of that determined in the presence of the unsaturated lipid substrate C₅₅-P. The purified MraY enzyme was also shown to accept both the reduced and unsaturated forms of C₅₅-P (unpublished data).

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FIG. 3. Specificity of the WecA enzyme for the lipid substrate. The importance of the carbon chain length of the lipid substrate for the activity of WecA was determined. A standard assay was carried out as detailed in the text using a 1.1 mM concentration of the tested lipid substrate. An activity value of 100% corresponds to 306 nmol/min/mg of protein. Data represent the mean of independent triplicate data sets, and the standard deviation was less than 10%.

The specificity of the WecA enzyme for the nucleotide substrate was also investigated to some extent. This enzyme did not accept UDP-MurNAc-pentapeptide, the nucleotide precursor of peptidoglycan biosynthesis, as a substrate. As we previously showed that UDP-GlcNAc was not a substrate for the MraY enzyme (6), these two paralogue enzymes clearly do not exhibit overlapping specificities and are specific to different biosynthesis pathways of the bacterial cell wall.

Effect of tunicamycin on WecA transferase activity. Tunicamycin is known to inhibit enzymes catalyzing the transfer of GlcNAc or MurNAc-pentapeptide motifs onto polyprenyl phosphate carrier lipids (14, 17). It was earlier shown to inhibit the pure MraY enzyme with a 50% inhibitory concentration of 12 µM (6). The WecA transferase activity was clearly inhibited by tunicamycin, and a 50% inhibitory concentration of 11 nM was determined (Fig. 4). Thus, this inhibitor was much more efficient (3 orders of magnitude) on WecA than on MraY.

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FIG. 4. Inhibition of WecA activity by tunicamycin. Incubation was performed as described in Materials and Methods except that various amounts of tunicamycin were added in the reaction mixture. Data represent the mean of independent triplicate data sets, and the standard deviation was less than 7%.

Conclusions. The purification to near homogeneity of a WecA transferase is described for the first time in this study. It allowed us to analyze the kinetic properties of this integral membrane enzyme in the absence of any contaminating protein (in particular, MraY and/or MurG) or C₅₅-P substrate originating from membranes. Its apparent stability and availability in milligram quantities now open the way to structural analyses of this membrane protein. The reproducibility of the purification procedure makes it well adapted for the purification of multiple mutants and the development of structure-activity relationship analyses. All of the available information in this present report will be useful in future work aimed at further molecular and functional characterization of this member of the polyprenyl-phosphate N-acetylhexosamine-1-phosphate transferase superfamily. The availability of pure WecA can be useful in the screening and search for inhibitors that could be exploited as antibacterial agents. Experiments concerning the enzymatic synthesis and purification of the WecA lipid product using the purified enzyme are currently under way in our laboratory. This will allow us to characterize the reverse reaction of WecA and its eukaryotic paralogue (GPT) as well as to study the reactions catalyzed by the enzymes catalyzing the subsequent steps in the respective biosynthesis pathways.

 
This work was supported by the Centre National de la Recherche Scientifique (UMR 8619) and by the European Community (EUR-INTAFAR, LSHM-CT-2004-512138). Bayan Al-Dabbagh is a recipient of a scholarship from the CROUS de Versailles, Service des Relations Internationales, Antony, France.

 
* Corresponding author. Mailing address: Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, Bât. 430, 91405 Orsay Cedex, France. Phone: 33 1 69 15 61 34. Fax: 33 1 69 85 37 15. E-mail: ahmed.bouhss{at}u-psud.fr

Published ahead of print on 22 August 2008.

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1
1. Alexander, D. C., and M. A. Valvano. 1994. Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. J. Bacteriol. 176:7079-7084.[Abstract/Free Full Text]
2
2. Amer, A. O., and M. A. Valvano. 2001. Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine. Microbiology 147:3015-3025.[Abstract/Free Full Text]
3
3. Amer, A. O., and M. A. Valvano. 2002. Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli. Microbiology 148:571-582.[Abstract/Free Full Text]
4
4. Amer, A. O., and M. A. Valvano. 2000. The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion. J. Bacteriol. 182:498-503.[Abstract/Free Full Text]
5
5. Anderson, M. S., S. S. Eveland, and N. P. Price. 2000. Conserved cytoplasmic motifs that distinguish sub-groups of the polyprenol phosphate:N-acetylhexosamine-1-phosphate transferase family. FEMS Microbiol. Lett. 191:169-175.[CrossRef][Medline]
6
6. Bouhss, A., M. Crouvoisier, D. Blanot, and D. Mengin-Lecreulx. 2004. Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis. J. Biol. Chem. 279:29974-29980.[Abstract/Free Full Text]
7
7. Bouhss, A., D. Mengin-Lecreulx, D. Le Beller, and J. Van Heijenoort. 1999. Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis. Mol. Microbiol. 34:576-585.[CrossRef][Medline]
8
8. Bouhss, A., A. E. Trunkfield, T. D. Bugg, and D. Mengin-Lecreulx. 2008. The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS Microbiol. Rev. 32:208-233.[CrossRef][Medline]
9
9. Burda, P., and M. Aebi. 1999. The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta 1426:239-257.[Medline]
10
10. Caravano, A., D. Mengin-Lecreulx, J. M. Brondello, S. P. Vincent, and P. Sinay. 2003. Synthesis and inhibition properties of conformational probes for the mutase-catalyzed UDP-galactopyranose/furanose interconversion. Chem. Eur. J. 9:5888-5898.[CrossRef]
11
11. Crouvoisier, M., D. Mengin-Lecreulx, and J. van Heijenoort. 1999. UDP-N-acetylglucosamine:N-acetylmuramoyl-(pentapeptide) pyrophosphoryl undecaprenol N-acetylglucosamine transferase from Escherichia coli: overproduction, solubilization, and purification. FEBS Lett. 449:289-292.[CrossRef][Medline]
12
12. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23-28.[CrossRef][Medline]
13
13. Dal Nogare, A. R., N. Dan, and M. A. Lehrman. 1998. Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase. Glycobiology 8:625-632.[Abstract/Free Full Text]
14
14. Elbein, A. D., J. Gafford, and M. S. Kang. 1979. Inhibition of lipid-linked saccharide synthesis: comparison of tunicamycin, streptovirudin, and antibiotic 24010. Arch. Biochem. Biophys. 196:311-318.[CrossRef][Medline]
15
15. El Ghachi, M., A. Bouhss, D. Blanot, and D. Mengin-Lecreulx. 2004. The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity. J. Biol. Chem. 279:30106-30113.[Abstract/Free Full Text]
16
16. Guo, H., W. Yi, J. K. Song, and P. G. Wang. 2008. Current understanding on biosynthesis of microbial polysaccharides. Curr. Top. Med. Chem. 8:141-151.[CrossRef][Medline]
17
17. Heifetz, A., R. W. Keenan, and A. D. Elbein. 1979. Mechanism of action of tunicamycin on the UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Biochemistry 18:2186-2192.[CrossRef][Medline]
18
18. Lehrer, J., K. A. Vigeant, L. D. Tatar, and M. A. Valvano. 2007. Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide. J. Bacteriol. 189:2618-2628.[Abstract/Free Full Text]
19
19. Lehrman, M. A. 1994. A family of UDP-GlcNAc/MurNAc:polyisoprenol-P GlcNAc/MurNAc-1-P transferases. Glycobiology 4:768-771.[Free Full Text]
20
20. Liu, J., and A. Mushegian. 2003. Three monophyletic superfamilies account for the majority of the known glycosyltransferases. Protein Sci. 12:1418-1431.[CrossRef][Medline]
21
21. McNeil, M. 1999. Arabinogalactan in mycobacteria: structure, biosynthesis and genetics, p. 207-223. In J. B. Goldberg (ed.), Genetics of bacterial polysaccharides. CRC Press, Boca Raton, FL.
22
22. Meier-Dieter, U., R. Starman, K. Barr, H. Mayer, and P. D. Rick. 1990. Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis. J. Biol. Chem. 265:13490-13497.[Abstract/Free Full Text]
23
23. Mikusova, K., M. Belanova, J. Kordulakova, K. Honda, M. R. McNeil, S. Mahapatra, D. C. Crick, and P. J. Brennan. 2006. Identification of a novel galactosyl transferase involved in biosynthesis of the mycobacterial cell wall. J. Bacteriol. 188:6592-6598.[Abstract/Free Full Text]
24
24. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
25
25. 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.[CrossRef][Medline]
26
26. Morona, R., C. Daniels, and L. Van Den Bosch. 2003. Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence. Microbiology 149:925-939.[Abstract/Free Full Text]
27
27. Pier, G. B. 2007. Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity. Int. J. Med. Microbiol. 297:277-295.[CrossRef][Medline]
28
28. Pigeon, R. P., and R. P. Silver. 1994. Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1. Mol. Microbiol. 14:871-881.[CrossRef][Medline]
29
29. Price, N. P., and F. A. Momany. 2005. Modeling bacterial UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. Glycobiology 15:29R-42R.[Abstract/Free Full Text]
30
30. Raymond, C. K., E. H. Sims, A. Kas, D. H. Spencer, T. V. Kutyavin, R. G. Ivey, Y. Zhou, R. Kaul, J. B. Clendenning, and M. V. Olson. 2002. Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa. J. Bacteriol. 184:3614-3622.[Abstract/Free Full Text]
31
31. Rush, J. S., P. D. Rick, and C. J. Waechter. 1997. Polyisoprenyl phosphate specificity of UDP-GlcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E. coli. Glycobiology 7:315-322.[Abstract/Free Full Text]
32
32. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
33
33. Soldo, B., V. Lazarevic, and D. Karamata. 2002. tagO is involved in the synthesis of all anionic cell-wall polymers in Bacillus subtilis 168. Microbiology 148:2079-2087.[Abstract/Free Full Text]
34
34. Valvano, M. A. 2003. Export of O-specific lipopolysaccharide. Front Biosci. 8:S452-S471.[CrossRef][Medline]
35
35. Whitfield, C. 1995. Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol. 3:178-185.[CrossRef][Medline]
36
36. Yamashita, Y., Y. Shibata, Y. Nakano, H. Tsuda, N. Kido, M. Ohta, and T. Koga. 1999. A novel gene required for rhamnose-glucose polysaccharide synthesis in Streptococcus mutans. J. Bacteriol. 181:6556-6559.[Abstract/Free Full Text]

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Journal of Bacteriology, November 2008, p. 7141-7146, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00676-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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