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Keywords:

  • UDP-N-acetylmuramic acid;
  • Peptidoglycan

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

UDP-N-acetylmuramic acid (UDP-MurNAc) is a precursor for peptidoglycan biosynthesis in bacteria. A major difficulty in the study of this pathway is that UDP-MurNAc is not commercially available. We have developed an enzymatic synthesis scheme for UDP-MurNAc using two easily purified Escherichia coli polyhistidine tagged peptidoglycan biosynthesis enzymes, MurZ and MurB, followed by a single-step purification of UDP-MurNAc by high-performance liquid chromatography. The identity of the UDP-MurNAc synthesized by our method was confirmed by electrospray ionization mass spectrometry. Furthermore, we show that the UDP-MurNAc can support a UDP-MurNAc-l-alanine ligase reaction.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Peptidoglycan is a structure unique to eubacteria and is responsible for maintaining cell shape, protecting the organism from osmotic pressure, and is involved in the processes leading to cellular division. Peptidoglycan fragments also possess many biological activities including pyrogenicity, toxigenicity, immunogenicity and somnogenicity [1–5]. The processes involved with the synthesis and assembly of the cell wall are targets for several classes of antibiotics.

The peptidoglycan is a network of interconnected polymers comprised of N-acetylglucosamine and N-acetylmuramic acid. Extending from the lactate group of N-acetylmuramic acid moieties are pentapeptides which become crosslinked by various transpeptidases (the penicillin binding proteins) once the peptidoglycan monomers become associated with the mature peptidoglycan layer outside of the cell. The formation of a cell wall can be divided into two phases: (1) intracellular synthesis of peptidoglycan monomers (N-acetylglucosamine-N-acetylmuramyl pentapeptide); (2) extracellular assembly of peptidoglycan monomers into a mature cell wall. Much research has been devoted to the characterization of the highly conserved enzymes involved with the intracellular stages of cell wall biosynthesis, also known as the Mur pathway [6,7].

Our laboratory is interested in studying the intracellular and extracellular peptidoglycan biosynthesis pathways of mycobacteria, areas of research that remain relatively unexplored. Our primary area of interest is the Mur pathway of mycobacteria.

With the exception of MurZ, whose substrate is UDP-N-acetylglucosamine (UDP-GlcNAc) [8], the cytoplasmic enzymes of the Mur pathway recognize peptidoglycan monomer intermediates containing UDP-N-acetylmuramic acid (UDP-MurNAc). Characterization of enzymes involved in these intracellular reactions requires the synthesis of intermediates starting from this basic building block. However, a major difficulty in the synthesis of these intermediates is that UDP-MurNAc is not commercially available. In bacteria, this nucleotide sugar is synthesized from UDP-GlcNAc by the enzymes MurZ and MurB [8–11]. We sought to develop a method to synthesize UDP-MurNAc for studies in our laboratory.

We decided to enzymatically synthesize UDP-MurNAc instead of using an older method of purifying it from cells [12]. Other groups have synthesized UDP-MurNAc enzymatically, but these studies used purified, wild-type enzyme and were not easily adaptable for a laboratory without experience in the synthesis of nucleotide sugars [9,11]. Therefore, we developed a straightforward enzymatic method for the synthesis of UDP-MurNAc using easily purified polyhistidine tagged Escherichia coli MurZ and MurB enzymes followed by a single-step high-performance liquid chromatography (HPLC) purification. This method allows for the relatively rapid synthesis and purification of UDP-MurNAc which will be useful in the study of the enzymes involved with peptidoglycan biosynthesis.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strains, culture techniques, reagents, and transformation of E. coli

All reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified. All bacterial strains used in this study are listed in Table 1. E. coli was grown in Luria–Bertani (LB) broth or on LB agar. All strains were incubated at 37°C. When necessary ampicillin was used at a final concentration of 100 μg ml−1 and isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. E. coli was made competent using a RbCl protocol [13]. For transformation, 1 μg plasmid DNA was added to 200 μl RbCl competent E. coli on ice for 30 min. Plasmid DNA uptake was initiated by heat shock at 42°C for 3 min. Cells were incubated at 37°C with shaking for 30 min in 1 ml of LB medium and plated on the appropriate media.

Table 1.  Strains used in this study
StrainDescriptionReference or source
E. coli K-12 HB101FΔ(gpt-proA)62 leuB1 glnV44 ara-14 galK2 lacY1 hsdS20 rpsL20 xyl-5 mtl-1 recA13[18]
E. coli K-12 DH10BFmcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 endA1 araΔ139 Δ(ara,leu)7697 galU galKλ-rpsL (Strr)Invitrogen
E. coli K-12 STBL2FmcrAΔ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96 supE44 relA1λ-Δ(lac-proAB)Invitogen
PM460E. coli STBL2/pMP146This study
PM461E. coli STBL2/pMP147This study
PM639E. coli DH10B/pMP198This study

2.2Plasmid construction

Plasmids used in this study are listed in Table 2. Plasmids pMP146 and pMP147 were constructed in E. coli STBL2, whereas pMP198 was constructed in E. coli DH10B. Plasmid DNA was purified using Qiagen columns (Qiagen Inc., Chatsworth, CA, USA) and DNA fragments were isolated using Gene Clean (Bio101, Vista, CA, USA). The wild-type murZ, murB, and murC genes were amplified from E. coli HB101 genomic DNA using Vent polymerase (NEB, Beverly, MA, USA). Oligonucleotides used for DNA amplification were synthesized by The University of Rochester Functional Genomics Center (Rochester, NY, USA) (Pv77, 78, 79, 80) and Invitrogen (Carlsbad, CA, USA) (Pv147, 149). The primers used for murZ amplification were Pv77 (5′-ggttcaagatctgataaatttcgtgttcagg-3′), Pv78 (5′-tcgacagaattcagttgatgcgtag-3′); murB Pv79 (5′-ggttcaagatctaaccactccttaaaaccctgg-3′), Pv80 (5′-agagtggaattcaccgttcgctaacagg-3′); and murC Pv147 (5′-atgtgcctcgagaatacacaacaattggcaaaactgcgttc-3′), Pv149(5′-cagaattcagagaagcttcccgctca-3′). Underlined sequences denote engineered restriction endonuclease sites used for cloning into the pTrcHis expression vectors (Invitrogen, Carlsbad, CA, USA) (Pv77 BglII, Pv78 EcoRI, Pv79 BglII, Pv80 EcoRI, Pv147 XhoI, Pv149 HindIII). All primers were based upon reported E. coli genomic DNA sequences: murZ (accession number M92358) [8], murB (accession number AAA24185) [10], murC (accession number AAB60787) [14]. Amplification reaction mixtures contained 100 ng of HB101 genomic DNA, 50 pmol of each primer, dNTPs at a concentration of 0.2 mM each, and 2 mM MgSO4. Reactions were carried out in a Perkin Elmer 2400 cycler (Norwalk, CT, USA) with the following parameters: 94°C for 5 min (1 cycle) and 94°C for 1 min, 55°C for 1.5 min, 72°C for 1.5 min (35 cycles). The amplified products were cloned into the appropriate pTrcHis expression vector. Plasmids were confirmed by restriction mapping and sequencing by ACGT (Northbrook, IL, USA) and The University of Rochester Functional Genomics Center (Rochester, NY, USA).

Table 2.  Plasmids used in this study
PlasmidDescriptionReference or source
pTrcHisAAmpr, pBR322 ori, lacIq, lacO, Ptrc, g10RBS, 6×histidine fusion vectorInvitrogen
pTrcHisBAmpr, pBR322 ori, lacIq, lacO, Ptrc, g10RBS, 6×histidine fusion vectorInvitrogen
pMP146pTrcHisB containing E. coli HB101 murZThis study
pMP147pTrcHisB containing E. coli HB101 murBThis study
pMP198pTrcHisA containing E. coli HB101 murCThis study

2.3Expression and purification of polyhistidine tagged MurZ, MurB, and MurC

Recombinant polyhistidine MurZ, MurB, and MurC proteins were batch purified under native conditions using Qiagen Ni-NTA resin (Qiagen Inc., Chatsworth, CA, USA). E. coli harboring pMP146, pMP147, or pMP198 from plates less than 1 week old, were inoculated into 100 ml LB broth containing ampicillin and IPTG and grown overnight at 37°C with shaking. The following morning cells were harvested at 3000×g and washed in ice-cold 50 mM Tris pH 8.0. The washed cells were centrifuged and pellets resuspended in 3 ml lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole) containing DNase and RNase at a final concentration of 100 μg ml−1. Cell suspensions were lysed in a French pressure cell (Aminco, Urbana, IL, USA) twice at 15 000 psi and cellular debris pelleted at 10 000×g. The remaining soluble cell extracts (∼3 ml) were added to 750 μl of a 50% Ni-NTA resin slurry (Qiagen Inc., Chatsworth, CA, USA) (aq) and gently rocked at 4°C for 1 h. The lysate/resin slurry was centrifuged (3000×g) at 4°C for 1 min and washed twice with 3 ml wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole). Recombinant polyhistidine tagged MurZ, MurB, and MurC proteins were eluted from the Ni-NTA resin three times with 300 μl of elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 250 mM imidazole). The purity and yield of the recombinant proteins were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the Bradford assay (BioRad, Hercules, CA, USA) respectively.

2.4MurZ and coupled MurZ/MurB assays

MurZ reaction mixtures contained UDP-GlcNAc (20 mM), phosphoenolpyruvate (PEP) (20 mM), dithiothreitol (DTT) (0.5 mM), Tris pH 8.0 (50 mM), and varying amounts of purified recombinant MurZ fusion protein ranging from 1 to 10 μg in a 50 μl reaction volume. Reactions were incubated at 37°C for 45 min. The release of inorganic phosphate from PEP was measured as previously described [15] with a few modifications; 10 μl aliquots of the reaction mixture were added to 800 μl of a 3:1 mixture of 0.045% malachite green (aq):4.2% ammonium molybdate in 4 N HCl. This mixture was incubated at room temperature for 30 min at which time the optical density at 660 nm was recorded.

Coupled MurZ and MurB reaction mixtures for the synthesis of UDP-MurNAc contained the following components in a final volume of 25 ml: UDP-GlcNAc (14 mM), PEP (14 mM), β-NADPH (14 mM), DTT (1 mM), Tris pH 8.0 (50 mM). Mixtures were prewarmed to 37°C and synthesis was initiated by the addition of 5 mg recombinant MurZ and 2.5 mg recombinant MurB polyhistidine tagged fusion proteins. Reactions were incubated at 37°C overnight. Control reactions lacked polyhistidine MurB fusion protein or PEP.

2.5Purification and quantitation of UDP-MurNAc

Protein was removed from the MurZ/B coupled UDP-MurNAc synthesis reaction using an Amicon Centriplus YM-10 filter device (Millipore Corp., Bedford, MA, USA), which reduced the reaction volume to 20.5 ml. Reaction components were separated by HPLC using a Waters 2695 separation module (Waters Corp., Milford, MA, USA) with a Waters μBondapack C18 column (3.9×150 mm, 10 μM pore size). Samples (10 μl) were injected into the column with an isocratic flow of 50 mM ammonium formate pH 3.5 at 1 ml min−1. Separations were performed at 30°C for 10 min and products were detected at 262 nm. Peaks containing UDP-MurNAc from 120 injections were pooled, lyophilized and dissolved in 500 μl of water.

The HPLC purified UDP-MurNAc was quantitated using a muramic acid assay that detects the presence of lactate released by alkali treatment of nucleotide sugar samples [16]. Purified UDP-MurNAc was diluted 1:10 in distilled water and aliquots ranging from 1 μl to 5 μl were adjusted to a final volume of 100 μl in water. 50 μl of 1 N NaOH was added to each sample and incubated at 37°C for 30 min. Following the alkaline incubation, 1 ml of concentrated H2SO4 was added and each sample placed into boiling water for 10 min. After cooling, 10 μl of 4% CuSO4 (aq) and 20 μl of a 1.5%p-hydroxydiphenyl solution in 95% ethanol were added and the reactions incubated at 30°C for 30 min at which time the optical density at 560 nm was recorded. The concentration of UDP-MurNAc was calculated by comparison to reactions performed with a lactate standard.

2.6Synthesis and purification of UDP-MurNAc-l-alanine

Reaction mixtures contained the following components in a final volume of 100 μl: UDP-MurNAc (0.5mM), l-alanine (0.5 mM), ATP (1 mM), β-mercaptoethanol (2.5 mM), MgCl2 (20 mM), (NH4)2SO4 (25 mM), Tris pH 7.5 (100 mM). Mixtures were prewarmed to 37°C and synthesis was initiated by the addition of 100 μg recombinant polyhistidine MurC fusion protein. Reactions were incubated overnight at 37°C. Control reactions lacked l-alanine or the polyhistidine MurC fusion enzyme.

Protein was removed from the MurC reaction using a Microsep 10 K Omega centrifugal filtration device (Pall Life Sciences, Ann Arbor, MI, USA). Reaction components were separated by HPLC using a Waters 2695 separation module with a μBondapack C18 column (3.9×150 mm, 10 μm pore size). Ten μl samples were injected into the column using an isocratic flow of 50 mM ammonium formate pH 3.5 at 1 ml min−1. Separations were performed at 25°C for 15 min. Nucleotide sugars and ADP/ATP were detected at 262 nm. Peaks containing UDP-MurNAc-l-alanine from 10 injections were pooled, lyophilized, and dissolved in an appropriate volume of water.

2.7Electrospray ionization mass spectrometry (ESI-MS) of purified sugar nucleotides

ESI-MS spectra were obtained in positive- and negative-ion detection modes using an Agilent 1100 Series LC/MSD Trap mass spectrometer. Samples were introduced in water containing 5% acetonitrile at a flow rate of 1 ml min−1. The in-line diode array detector was set to monitor optical density at 220–450 nm and store signals at 210 and 260 nm. Nitrogen was used as the drying gas at 11 l min−1 and 350°C at a nebulizer pressure of 60 psig. The scan range was 100–800 m/z using five averages and 13 000 m/z s−1 resolution. The capillary voltage was −4000 V for positive-ion detection and +4000 V for negative ions, with an endplate offset of −500 V. Processing was done off-line using HPChemstation.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Purification of E. coli polyhistidine tagged MurZ, MurB, and MurC recombinant proteins

The E. coli murZ, murB, and murC genes were amplified from E. coli HB101 genomic DNA with sequence specific primers using the PCR. The amplified genes were cloned into the expression vector pTrcHisB (murZ and murB) and pTrcHisA (murC), which fused a tag of six histidine residues onto the amino termini of the proteins. The murZ (pMP146), murB (pMP147), and murC (pMP198) constructs were expressed in E. coli and analysis of extracts from these clones confirmed the production of three proteins with molecular masses similar to that of the expected size of the fusion proteins (Fig. 1). Each of these fusion proteins were easily purified to a high degree of homogeneity by nickel affinity chromatography (Fig. 1). Purified, active MurB (a flavoprotein) was visually identified by a bright yellow color.

image

Figure 1. Purification of polyhistidine tagged MurZ, MurB, and MurC fusion proteins. Coomassie stained SDS–PAGE gel of whole cell extract (X), unbound fraction (U), washes (W1, W2) and eluate (E) from a native small-scale batch purification of E. coli carrying (A) pMP146 (murZ), (B) pMP147 (murB), and (C) pMP198 (murC).

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3.2The polyhistidine tagged MurZ and MurB fusion proteins are active

MurZ is an UDP-GlcNAc-enolpyruvyl transferase which converts UDP-GlcNAc to UDP-GlcNAc-enolpyruvate in a PEP dependent reaction [8]. To test the enzymatic activity of the MurZ fusion protein we used a phosphate release assay to monitor the liberation of inorganic phosphate from PEP (see Section 2). Increasing the amount of purified MurZ enzyme in the reactions resulted in a concomitant increase of inorganic phosphate release from PEP. A reaction containing 10 μg of MurZ enzyme resulted in phosphate release 25-fold greater than that of a control reaction lacking the fusion protein (data not shown). As expected, the addition of the MurZ specific inhibitor phosphomycin to reactions containing 10 μg of recombinant MurZ abrogated inorganic phosphate release (data not shown). MurB, a NADPH dependent UDP-GlcNAc-enolpyruvate reductase [10], was assayed by monitoring the decrease in optical density at 340 nm as NADPH was oxidized to NADP+ in a coupled reaction consisting of recombinant MurZ, PEP, and UDP-GlcNAc [9]. The enzymatic activity of a reaction containing all components was 60-fold greater than control assays lacking either recombinant MurB or PEP (data not shown).

3.3Synthesis and purification of UDP-MurNAc

UDP-MurNAc was synthesized from UDP-GlcNAc using purified polyhistidine MurZ/B fusion proteins and analyzed by HPLC following filtration of the coupled reaction mixture. Three nucleotide sugar species were detected upon separation by HPLC (Fig. 2); the substrate UDP-GlcNAc, the intermediate UDP-GlcNAc-enolpyruvate, and the product UDP-MurNAc. The UDP-MurNAc peak eluted from the column between 2.6 and 3.4 min (Fig. 2) and was not observed in a control reaction which lacked polyhistidine tagged MurB or PEP (data not shown). Fractions containing UDP-MurNAc were pooled, lyophilized, and concentrated. HPLC of purified UDP-MurNAc is shown in Fig. 3. UDP-MurNAc was quantitated by assaying for lactate (see Section 2). We recovered 11.5 to 15 μmol of UDP-MurNAc from 1.2 ml aliquots of the coupled reaction mixture. While the MurZ/MurB reaction appears to be nearly complete, our recovery of product is 60–73% relative to the starting material.

image

Figure 2. UDP-MurNAc identification by HPLC. UDP-MurNAc was separated from the MurZ/B coupled reaction components on a C18 column using reverse phase HPLC. Nucleotide sugars were detected at a wavelength of 262 nm. The fractions from 2.6 to 3.4 min were pooled and lyophilized for further analyses.

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image

Figure 3. HPLC purified UDP-MurNAc. Following separation of MurZ/B reaction components the peaks containing UDP-MurNAc were collected and analyzed by HPLC to confirm purity.

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3.4Synthesis of UDP-MurNAc-l-alanine

We used our purified UDP-MurNAc in a MurC reaction to demonstrate that our product can support an enzymatic reaction. MurC is a UDP-MurNAc-l-alanine ligase in the Mur pathway which converts UDP-MurNAc to UDP-MurNAc-l-alanine in an ATP and l-alanine dependent reaction [14]. The MurC reaction components were separated by HPLC and a unique species was observed with a retention time of 7.2 min (Fig. 4) that was not present in control reactions that were devoid of recombinant MurC or l-alanine (data not shown). The addition of an amino acid containing a non-polar side group, such as l-alanine, to UDP-MurNAc will presumably result in an increased retention rate of UDP-MurNAc-l-alanine in relation to UDP-MurNAc under our HPLC conditions. As expected, an increased retention time was observed for the product of the MurC reaction.

image

Figure 4. HPLC separation of the MurC reaction components. The UDP-MurNAc-l-alanine peak from 6.6 to 7.9 min was collected and lyophilized for further analysis.

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3.5ESI-MS analysis of UDP-MurNAc and UDP-N-acetylmuramyl-l-alanine

The sugar nucleotide products from the enzymatic reactions above were further characterized by ESI-MS. We initially examined UDP-GlcNAc as a model compound. In positive-ion detection mode this was characterized by an [M+H]+ ion at m/z 608 and a prominent base peak for the GlcNAc oxonium ion at 204. Carbohydrate oxonium ions are typically observed for glycosidic bond cleavage [17]. In negative-ion detection mode an identifying base peak was observed for the molecular ion [M−H] at m/z 606, plus a minor peak at m/z 444. Structurally, UDP-MurNAc differs from UDP-GlcNAc by the ether linked pyruvic acid group attached to position 3 of the GlcNAc ring. Hence the mass of UDP-MurNAc is hypothetical 72 mass units greater than UDP-GlcNAc. The expected [M+H]+ ion was observed for UDP-MurNAc at m/z 680, with the accompanying MurNAc oxonium ion at m/z 276 (Fig. 5A, left). Negative-ion detection identified the predicted peak for [M−H] at m/z 678 (Fig. 5A, right). Similarly, the addition of the alanine residue to UDP-MurNAc to generate UDP-MurNAc-l-alanine increases its relative mass by 71 mass units, from 679 to 750. The predicted [M+H]+ for UDP-MurNAc-l-alanine was apparent at m/z 751 (Fig. 5B, left) and the MurNAc-l-alanine oxonium fragment ion at m/z 347 (Fig. 5B, left). Examination of the UDP-MurNAc-l-alanine in negative-ion detection mode identified the expected peak for [M−H] at m/z 749 (Fig. 5B, right). Hence, analysis by ESI-MS confirms the identity of the enzymatic products as UDP-MurNAc and UDP-MurNAc-l-alanine.

image

Figure 5. UDP-MurNAc and UDP-MurNAc-l-alanine ESI-MS. Positive-ion detected ESI-MS spectra (left) and negative-ion detected ESI-MS spectra (right) for UDP-MurNAc (A) and UDP-MurNAc-l-alanine (B).

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Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by a grant from the National Institutes of Health (AI4731) and a Burroughs Wellcome Career Award in the Biomedical Sciences to M.S.P.; J.B.R. is supported by the Molecular Pathogenesis of Bacteria and Viruses Training Grant AI07362.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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