From the
Department of Biochemistry, Duke University Medical Center and the
Department of Radiology and the Duke NMR Spectroscopy Center, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, April 17, 2003
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ABSTRACT |
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INTRODUCTION |
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The Kdo-lipid A portion of LPS, which is relatively conserved among diverse Gram-negative bacteria, is sufficient to support the growth of Escherichia coli or Salmonella typhimurium (2, 8). Certain covalent modifications to Kdo-lipid A are induced by environmental stimuli, such as low magnesium ion concentrations and/or low pH, as would be encountered by bacteria in phagolysosomes of macrophages (911). As shown in Fig. 1 for S. typhimurium, these modifications include the incorporation of palmitate (12, 13), the formation of S-2-hydroxymyristate (14), the addition of phosphoethanolamine (pEtN) (15), and/or the addition 4-amino-4-deoxy-L-arabinose (L-Ara4N) moieties (Fig. 1) (1517). Addition of the L-Ara4N and pEtN moieties to the phosphate groups of lipid A confers resistance to polymyxin and various cationic antimicrobial peptides (1517). Resistance is thought to occur by reduction of the net negative charge of lipid A, resulting in a reduced affinity for cationic peptides or polymyxin, thereby preventing these substances from penetrating the outer membrane.
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The L-Ara4N modification of lipid A is governed by the PmrA/PmrB two-component regulatory system (10, 1821), which is switched on by low pH. The transcription factor PmrA activates genes at the pmrE(ugd) and pmrHFIJKLM loci (10, 22). Constitutive mutants of pmrA (pmrAc) have been isolated in E. coli and S. typhimurium; these mutants modify their lipid A with L-Ara4N and pEtN groups under all growth conditions, and they are polymyxin-resistant (17, 20, 23). However, inactivation of pmrE or of any of the genes in the pmrHFIJKL operon in a pmrAc S. typhimurium mutant results in loss of polymyxin resistance and loss of the L-Ara4N modification of lipid A (10).
Sequence analyses of the various genes in the pmr loci have led us to propose a pathway for the biosynthesis of the L-Ara4N moiety and its attachment to lipid A (22). Our hypothesis (Fig. 2) is supported by the following observations. 1) ArnT (PmrK) is an inner membrane enzyme that utilizes the novel glycolipid undecaprenyl phosphate--L-Ara4N as the donor of the L-Ara4N moiety to modify lipid A (16, 17). 2) ArnA (PmrI) is a dehydrogenase/decarboxylase that converts UDP-glucuronic acid to a novel 4''-keto-pentose, designated UDP-Ara4O, which can be isolated in milligram quantities as the hydrated ketone (22). 3) Cell extracts of polymyxin-resistant (but not polymyxin-sensitive) E. coli cells convert UDP-Ara4O to the putative sugar nucleotide UDP-L-Ara4N in the presence of L-glutamate (22). 4) Cell extracts of polymyxin-resistant E. coli cells can further convert the proposed UDP-L-Ara4N to a putative formyl-amino derivative in the presence of N-10-formyltetrahydrofolate (22).
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We have now cloned, overexpressed, and purified to homogeneity the aminotransferase ArnB (PmrH). We demonstrate that ArnB (GenBankTM accession number AAM92146
[GenBank]
) contains a pyridoxal phosphate cofactor and catalyzes the reversible transfer of the -amine moiety from L-glutamate to the C-4'' position of UDP-Ara4O, generating UDP-L-Ara4N. The structure of UDP-L-Ara4N was validated by one- and two-dimensional NMR spectroscopy. The transamination is highly specific for glutamate as the amine donor. Our biochemical data establish ArnB as the key aminotransferase required for the biosynthesis of the L-Ara4N moiety present on lipid A molecules of polymyxin-resistant Gram-negative bacteria. Obvious ArnB orthologs are present in Yersinia pestis, Pseudomonas aeruginosa, and Burkholderia cepacia, all of which are capable of modifying their lipid A with L-Ara4N (24). The purification of ArnB permits the enzymatic synthesis of milligram quantities of UDP-L-Ara4N, which can now be used to probe for the remaining steps in the transfer of L-Ara4N to lipid A. A crystal structure of ArnB (25), recently obtained as part of a structural genomics program, is in accord with our enzymatic and biochemical studies.
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EXPERIMENTAL PROCEDURES |
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Strains and Recombinant DNA TechniquesConstruction of WD101, a polymyxin-resistant mutant of E. coli W3110, was described previously (17). Preparation and transformation of competent cells by the CaCl2 method, genomic DNA purification, and gel electrophoresis of restriction fragments were performed according to published procedures (27, 28). Plasmids were purified using the QIAprep spin miniprep kit (Qiagen). Restriction enzymes, Pfu DNA polymerase, and T4 DNA ligase were used as recommended by their manufacturers. DNA sequencing was performed on an ABIprism 377 instrument at the Duke University DNA Analysis Facility.
Construction of pETArnB, pETArnB-C6H, and pETArnB-N6HThe predicted coding region for ArnB (GenBankTM accession number AAM92146 [GenBank] ) was amplified by PCR from E. coli W3110 genomic DNA with primers to the arnB 5' end (5'-CGGGATCCATGGCGGAAGGAAAAGCAATG-3') and to the arnB 3' end (5'-CGGGATCCTCGAGTTATTGTCCTGCGAGTTGCTG-3'), containing engineered NcoI and XhoI restriction sites, respectively. The Opti-PrimeTM PCR Optimization kit (Stratagene) was used as described by the manufacturer. The amplification reaction (50 µl) contained the following components: 5 µl of 10x Opti-Prime Buffer 9 (100 mM Tris-HCl, pH 9.2, 15 mM MgCl2, 250 mM KCl), 0.2 mM of each dNTP, 250 ng of genomic DNA, 0.5 µM of each primer, and 2.5 units of Pfu DNA polymerase. After 30 cycles (94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1.5 min) followed by 1 cycle (94 °C for 1 min, 50 °C for 1 min, and 72 °C for 10 min), a single product of the predicted size was obtained. This was isolated with the QIAquick PCR Purification kit (Qiagen), digested with XhoI and NcoI, and then ligated into the T7lac expression vector, pET28b (Novagen), previously digested with the same enzymes. The presence of the arnB gene (29) and appropriate flanking DNA was confirmed by sequencing of final construct, which was designated pETArnB.
Amplification of DNA for cloning a modified arnB gene expressing a C-terminal hexa-histidine fusion protein of ArnB (designated ArnB-C6H) utilized the arnB 5' primer described above for the native protein and the oligonucleotide 5'-CGGGATCCTCGAGTTGTCCTGCGAGTTGCTGAAG-3' as the 3' primer. The latter contained an engineered XhoI restriction site. The PCR (50 µl) contained the following: 5 µl of 10x Opti-Prime Buffer 11 (100 mM Tris-HCl, pH 9.2, 35 mM MgCl2, 250 mM KCl), 0.2 mM of each dNTP, 250 ng of genomic DNA, 0.5 µM of each primer, and 2.5 units of Pfu DNA polymerase. The same temperature parameters described above were used for the PCR. The resulting product was cloned into the expression vector pET28b, as described above, to generate pETArnB-C6H. The final construct was confirmed by DNA sequencing.
Amplification of DNA for cloning a modified arnB gene encoding an N-terminal hexa-histidine fusion protein of ArnB (designated ArnB-N6H) was accomplished using the same reaction conditions as for pETArnB-C6H. The arnB 3' primer described above for the native protein was used together with the oligonucleotide 5'-CGGATCCATATGGCGGAAGGAAAAGCAATG-3' as the 5' primer, which also contained an engineered NdeI restriction site. The PCR product was isolated with the QIAquick PCR Purification kit (Qiagen), digested with XhoI and NdeI, and ligated into the pET28b expression vector, previously digested with the same enzymes, to generate pETArnB-N6H. The final construct was confirmed by DNA sequencing.
Overexpression of the arnB Gene Product and Purification of ArnB-N6H and ArnB-C6HPlasmids pETArnB, pETArnB-N6H, or pETArnB-C6H were transformed into E. coli NovaBlue(DE3) cells (Novagen). Cultures were grown overnight at 37 °C from single colonies in LB broth (30) supplemented with 30 µg/ml kanamycin. The overnight culture was then used to inoculate a fresh 100-ml culture of the same broth at A600 0.01. Cells were grown at 37 °C to A600 of
0.6. Expression of ArnB was induced by addition of isopropyl-
-D-thiogalactopyranoside to a final concentration of 1 mM, and growth was continued at 30 °C for 3.5 h. Cells were harvested by centrifugation at 6500 x g for 15 min at 4 °C. The cell pellet was resuspended in 3 ml of buffer A, consisting of 25 mM HEPES, pH 7.5, 10% glycerol, and 300 mM NaCl. Cells were broken by passage through a French pressure cell at 18,000 pounds/square inch. Cell debris was removed by centrifugation at 16,500 x g for 15 min at 4 °C to give crude cell-free extracts.
For the hexa-histidine fusion proteins, the crude cell-free extracts were loaded onto a 1-ml nickel-nitrilotriacetic acid column (Qiagen), which was pre-equilibrated in buffer A. The column was washed with 15 column volumes of buffer A and 15 column volumes of buffer A containing 50 mM imidazole; it was then eluted with 15 column volumes of buffer A containing 100 mM imidazole. Proteins were concentrated, and the imidazole was removed by exchanging for buffer B (consisting of 50 mM HEPES, pH 7.5, and 10% glycerol) with the use of a Centricon® (YM-10) membrane, which was subjected to several cycles of low speed centrifugation and dilution in buffer B. Protein concentrations were determined using the bicinchoninic acid procedure (26).
The overexpression of ArnB, ArnB-N6H, and ArnB-C6H, as well as the initial purification of the hexa-histidine fusion proteins, was monitored by SDS-PAGE (4% stacking gel and 8% resolving gel), followed by Coomassie Blue staining (31).
Enzymatic Synthesis and Isolation of UDP-L-Ara4NA 10-ml reaction mixture, containing 0.05 mg/ml pure ArnB-N6H, 1 mM UDP-Ara4O, and 10 mM L-glutamate in 50 mM HEPES, pH 7.5, was incubated at 30 °C for 5.5 h. Protein was removed by passing the reaction mixture through a Centricon® (YM-10) membrane. The enzymatic product was then loaded onto a 10-ml column of DEAE-SepharoseTM CL-6B, pre-equilibrated with 10 mM BisTris, pH 5.9. The column was washed with 50 ml of deionized water, followed by elution of the product with a 0100 mM BisTris, pH 5.9, gradient (200-ml total volume). Chromatography was monitored by absorbance of the effluent at 254 nm. Fractions (12 ml) containing the desired product, which eluted at about 30 mM BisTris, were pooled and diluted 10-fold with H2O. The diluted material was then loaded onto a 10-ml column of DEAE-SepharoseTM CL-6B, pre-equilibrated with 10 mM triethylammonium bicarbonate, pH 8.5. The column was washed with 50 ml of deionized water, followed by elution of the product with a 0100 mM gradient of triethylammonium bicarbonate, pH 8.5 (200-ml total volume). Chromatography was again monitored by absorbance at 254 nm. Fractions containing the product (which eluted at about 60 mM triethylammonium bicarbonate) were pooled and concentrated by lyophilization to give the triethylammonium salt of the sugar nucleotide. Conversion to the sodium salt was accomplished by passage over a 1-ml column of AG®50W-X8 resin (sodium form), followed by lyophilization to give the pure compound (1.7 mg, 31% yield).
Enzymatic Synthesis and Purification of UDP-L-[U-14C]Ara4NConversion of UDP-[U-14C]glucuronic acid (380 mCi/mmol) to UDP-L-[U-14C]Ara4N was performed in 400-µl reaction mixtures containing 50 mM HEPES, pH 7.5, 2 mM dithiothreitol, 3 mM NAD+, 1 mM L-glutamate, 0.05 mg/ml ArnA overproducing cell-free extract (22), 0.05 mg/ml pure ArnB-C6H, and 13 µM UDP-[U-14C]glucuronic acid (2 µCi), previously dried under nitrogen and re-dissolved in a small volume of deionized water. The reaction proceeded for 220 min at 30 °C and was then diluted to 1.5 ml with deionized water before loading onto a 2-ml column of DEAE-SepharoseTM CL-6B, pre-equilibrated with 10 mM BisTris, pH 6. The column was washed with 5 ml of deionized water, followed by elution of the nucleotides with a 0100 mM gradient of BisTris, pH 6 (40-ml total volume). Fractions containing UDP-L-[U-14C]Ara4N were pooled, diluted 10-fold with deionized water, and loaded onto a 2-ml column of DEAE-SepharoseTM CL-6B, pre-equilibrated with 10 mM triethylammonium bicarbonate, pH 8.5. The column was washed with 5 ml of deionized water. The NAD+ was resolved from the desired sugar nucleotide with a 0100 mM gradient of triethylammonium bicarbonate, pH 8.5 (40-ml total volume). The radioactive fractions were concentrated using a Speed-Vac centrifuge and dissolved in 250 µl of deionized water. The yield of UDP-L-[U-14C]Ara4N was 74% based on the input radioactivity. The product was stored frozen at 20 °C.
Nuclear Magnetic Resonance SpectroscopyThe sodium salt of the putative UDP-L-Ara4N (1.7 mg prepared as described above) was dissolved in 0.6 ml of 99% D2O and placed into a 5 mm NMR tube. Measurements with a 3-mm pH electrode revealed a pD value of 8.00. Proton and 13C NMR chemical shifts were referenced relative to 2,2-dimethylsilapentane-5-sulfonic acid at 0.00 ppm.
1H NMR spectra were obtained on a Varian Inova 800 spectrometer equipped with a Sun Ultra 10 computer and a 5-mm Varian triple resonance probe. 1H spectra were obtained with a spectral width of 8.0 kHz, a 76° pulse flip angle (7 µs), a 4.0-s acquisition time, and a 1.5-s relaxation delay, and were digitized using 96,000 points to obtain a digital resolution of 0.16 Hz/point. Two-dimensional NMR experiments were performed as described previously (17, 22, 32).
ArnB Enzymatic AssaysThe rate of formation of UDP-L-[U-14C]Ara4N by ArnB was quantified using a coupled assay in which overexpressed ArnA was first used to generate UDP-[U-14C]Ara4O in situ from UDP-[U-14C]glucuronic acid. The UDP-[U-14C]Ara4O is relatively unstable at the low chemical concentrations needed for radioactive assays. Briefly, the initial ArnA reaction mixture (10 µl) contained 90 mM HEPES, pH 7.5, 3 mM NAD+, and 300 µM UDP-[U-14C]glucuronic acid (0.02 µCi), and 0.1 mg/ml ArnA overproducing cell-free extract from E. coli NovaBlue(DE3)/pETArnA (22). Complete conversion of UDP-[U-14C]glucuronic acid to UDP-[U-14C]Ara4O was achieved after 120 min at 30 °C. To assay for UDP-L-[U-14C]Ara4N synthesis, the amine donor (usually glutamate) and ArnB were added to bring the final volume to 20 µl; this was set up to yield final concentrations of 50 mM HEPES, pH 7.5, 150 µM UDP-[U-14C]Ara4O (unless otherwise indicated), and 7.5 mM amine donor. Reactions were incubated further at 30 °C. At the desired time points, a 1-µl portion was withdrawn and spotted onto a polyethyleneimine-cellulose TLC plate. The spots were allowed to dry, and the plate was washed in methanol for 5 min. After drying, the plate was developed using 0.4 M LiCl in 0.25 M aqueous acetic acid. The plate was dried and exposed to a PhosphorImager screen. Remaining substrate and radioactive product were quantified with a PhosphorImager (ImageQuant software, Amersham Biosciences). ArnB specific enzymatic activity was calculated in units of nmol/min/mg.
Ultraviolet-visible Spectroscopy of ArnBPurified ArnB-C6H (25 µM)in50mM HEPES, pH 7.5, containing 10% glycerol was incubated at 30 °C alone or in the presence of 1 mM L-glutamate in a total volume of 100 µl. Spectra were recorded on a BeckmanCoulter DU® 640B UV-visible scanning spectrophotometer. Experimental data were exported into Kaleidagraph (Synergy Software, Reading, PA) and plotted. The reactions were blanked against 50 mM HEPES, pH 7.5, containing the buffer alone (10% glycerol in 50 mM HEPES, pH 7.5).
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RESULTS |
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Upstream of arnA resides arnB (pmrH), another gene required for polymyxin resistance in S. typhimurium (10), which is predicted to encode an aminotransferase (22, 33). The genetic location of arnB and the predicted sequence of its encoded protein made it a likely candidate for the proposed transaminase involved in the UDP-L-Ara4N formation (Fig. 2). To validate the function of ArnB, the full-length arnB gene was cloned into the expression vector pET28b behind an inducible T7lac promoter. Expression was carried out in E. coli NovaBlue(DE3), a polymyxin-sensitive strain that does not modify its endogenous lipid A with the L-Ara4N moiety (16). A protein of the predicted molecular mass of ArnB (43 kDa) was observed by SDS-gel electrophoresis in cell-free extracts of induced cells but was absent in uninduced controls (Fig. 3, lanes 2 and 3, respectively). Extracts of induced cells containing pETArnB catalyzed the rapid L-glutamate-dependent conversion of UDP-Ara4O to a species with the RF expected for UDP-L-Ara4N (data not shown).
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The relatively low level of ArnB expression, as judged by gel electrophoresis (Fig. 3), led us to engineer hexa-histidine sequences into the protein to facilitate purification. To this end, arnB was cloned into the pET28b expression vector to express either an N-terminal hexa-histidine fusion protein (ArnB-N6H) containing an additional 20 amino acids, or a C-terminal hexa-histidine fusion protein (ArnB-C6H) containing an additional 7 amino acids. Similar to the wild-type ArnB, the hexa-histidine fusion proteins were expressed in induced E. coli NovaBlue(DE3) but were absent in uninduced controls (Fig. 3, lanes 5 and 4 and lanes 8 and 9, respectively). The ArnB-specific activity of a cell extract of induced E. coli NovaBlue(DE3)/pETArnB-C6H is 130 nmol/min/mg. Approximately the same specific activity is seen with cell extracts overexpressing ArnB-N6H or wild-type ArnB. By using nickel chelation chromatography, the hexa-histidine ArnB fusion proteins were purified to homogeneity, as judged by SDS gel electrophoresis (Fig. 3, lanes 6 and 7). The final specific activity of the pure proteins is 1.3 x 103 nmol/min/mg.
Analysis of the Covalent Structure of the ArnB Product by One-dimensional 1H NMR SpectroscopyBy using pure ArnB-N6H, 10 µmol of UDP-Ara4O was converted to the putative UDP-L-Ara4N in the presence of 100 µmol of L-glutamate, as described under "Experimental Procedures." The substrate and product were separated using anion exchange chromatography at pH 5.9 (0100 mM BisTris). The fractions containing the ArnB product were then separated from the BisTris, using anion exchange chromatography at pH 8.5 in the volatile buffer, triethylammonium bicarbonate (0100 mM). Fractions containing the desired product were lyophilized to give the triethylammonium salt of the sugar nucleotide, which was converted to the sodium salt by passage through a Dowex AG®50W-X8 column (sodium form).
The sodium salt (1.7 mg) of the product was dissolved in D2O, and its 1H NMR spectrum was recorded, Fig. 4 (panels A and B). The uracil and ribose moieties of the substrate, UDP-Ara4O, and of the ArnB product possess virtually identical chemical shifts and coupling constants (Table I). However, the well resolved signals arising from the pyranose moiety of the UDP-Ara4O and the putative UDP-L-Ara4N show significant differences (Fig. 4, panels B versus C, and Table I). The one-dimensional 1H NMR analysis therefore suggests that ArnB alters only the pyranose moiety of UDP-Ara4O.
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Characterization of the ArnB Product by Two-dimensional 1H NMR SpectroscopyThe two-dimensional 1H-1H COSY NMR spectrum of the putative UDP-L-Ara4N product (Fig. 5) was used to assign the protons in the ribose, uracil, and pyranose rings. As suggested by the one-dimensional spectra, the uridine nucleoside moiety was not altered by ArnB (Table I). Close similarities between the putative UDP-L-Ara4N and the UDP-Ara4O spectra include the strong cross-peak connectivity between the uracil H-6 and H-5 doublet resonances (not shown), the H-1' connectivity to H-2' (4.39 ppm), the H-3' connectivity to H-4' (4.30 ppm), and the connectivities of H-4' to H-5b' and H-5a' (Table I).
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The assignments for the protons of the pyranose moiety of the ArnB product (Fig. 4) were unambiguously obtained from the COSY analysis (Fig. 5). The anomeric H-1'' proton signal, a double-doublet at 5.63 ppm (JH1'',2'' = 3.4, JH1'',P = 7.3 Hz), shows a cross-peak to the H-2'' double-triplet at 3.77 ppm J1'',2'' = 3.4, J2'',3'' = 9.9 Hz). A second cross-peak from H-2'' correlates to H-3'' at 4.15 ppm (dd, J2'',3'' = 9.9, J3'',4'' = 4.6 Hz). H-3'' then connects to H-4'' at 3.59 ppm (unresolved multiplet). Further tracing of the two remaining cross-peaks from H-4'' locate the H-5b'' (4.29 ppm; dd, J4'',5'' = 2.1, J5b'',5a'' = 13.3 Hz) and H-5a'' (3.80 ppm; dd, J4'',5'' = 2.0, J5a'',5b'' = 13.3 Hz) proton signals.2
The small J1'',2'' coupling (3.4 Hz) and the large J2'',3'' coupling (9.9 Hz) indicate that the pyranose ring is in a configuration with an equatorially disposed H-1'', and with axial H-2'' and H-3'' protons, which would be classified as the anomer, given the L stereochemistry of the sugar (34). The two-dimensional NOE spectroscopy data (not shown) revealed a strong intra-molecular cross-peak between H-1'' and H-2'', in accord with an equatorial H-1'' and axial H-2'' on the same face of the sugar (Fig. 2) (35). The large trans-diaxial J2'',3'' coupling then suggests that H-3'' is likewise axial. The intermediate value of the J3'',4'' coupling (4.6 Hz) indicates an axial-equatorial coupling, showing that H-4'' is in the equatorial position (Fig. 2). A strong intra-molecular cross-peak is observed (not shown) between H-3'' and H-4'' further confirming that they are on the same face (Fig. 2).
The measured values of 2.1 and 2.0 Hz for the two J4'',5'' couplings (equatorial-equatorial and equatorial-axial) do not allow distinction of the axial or equatorial H-5'' protons. Attempts to distinguish these protons for this pyranose ring directly through two-dimensional NOE experiments were unsuccessful, as no clear NOE cross-peak was observed between the axially disposed H-3'' (Fig. 2) and either H-5'' proton, at both 600- and 800-MHz fields. The equatorial H-4'' showed a stronger NOE cross-peak to H-5b'' (4.29-ppm signal) and a weaker NOE cross-peak to H-5a'' (3.80-ppm signal), but those results do not allow firm assignments. However, the H-5a'' (3.80 ppm) and H-5b'' (4.29 ppm) signals are assigned to the axial and equatorial positions following previous literature values with synthetic standards (34, 36), based upon the chemical shift consideration that a proton in an axial position at a given carbon atom on a pyranose ring is expected to resonate at
0.5 ppm higher field than the corresponding equatorial proton attached to the same carbon (37). Furthermore, the two-dimensional NOE spectroscopy data for the
-anomeric L-Ara4N ring of the natural product undecaprenyl phosphate-L-
-Ara4N (17) yielded the expected NOE cross-peaks between H-1'', H-3'' and the upfield H-5'' proton resonance, in agreement with the literature assignments, and distinctly different from what is observed with the ArnB product generated in vitro.
Evaluation of the Carbon Structure of the ArnB Product by HMQC/HMBC Spectroscopy13C NMR data for the ArnB enzymatic product were obtained indirectly through 1H-detected HMQC and HMBC two-dimensional NMR experiments. The partial two-dimensional HMQC 1H-13C correlation map (Fig. 6, panel A) reveals three direct 1H-13C single-bond correlations in the anomeric region (90105 ppm). The anomeric H-1'' near 5.63 ppm correlates to the carbon resonance of the pyranose at 98.2 ppm (C-1''). The C-1 atom of a pyranose ring that is glycosidically linked with an axial oxygen is expected to resonate in the 98103-ppm range, whereas a C-1 atom that is glycosidically linked with an equatorial oxygen is expected to resonate in the 103106-ppm region (35). The 98-ppm shift of C-1'' for the 4-amino-4-deoxy-arabinose is thus consistent with an axially disposed oxygen atom and an equatorially disposed H-1'', as deduced from the 1H NMR data. The ribose H-1' proton signal at 6.00 ppm correlates to the anomeric carbon resonance at 90.9 ppm (C-1'). The uracil H-5 near 5.99 ppm correlates to the 105.3-ppm carbon signal, and the uracil H-6 at 7.97 ppm correlates to a carbon signal at 144.2 ppm (not shown).
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Examination of the HMQC data for the other proton resonances of the pyranose moiety shows that the H-5b'' and H-5a'' proton multiplets correlate to a carbon signal at 62.4 ppm (C-5''), whereas the H-2'' and H-3'' multiplets connect to carbon resonances at 70.1 and 68.5 ppm, respectively. These carbon shift positions correspond to oxygen-substituted carbons of sugars. The nitrogen-substituted carbons of amino sugars are reported to resonate near 5255 ppm (35, 38). The H-4'' multiplet shows a prominent cross-peak near 55 ppm. This C-4'' chemical shift is diagnostic evidence confirming C-4'' as the site of the amino group substitution. The remaining sugar HMQC cross-peaks arise from protonated carbons of the ribose ring. The ribose assignments derived (Table I) agree with the literature values (35, 39).
The multibond HMBC two-dimensional map (Fig. 6, panel B) allows identification of the quaternary carbons leading to full assignments (Table I) for the enzyme product. Analyses of the multibond correlations from the uracil H-6 (7.97 ppm) and H-5 (5.99 ppm) yielded assignment of the 154 and 168 ppm quaternary carbon signals to C-2 and C-4, respectively (not shown in Fig. 6, panel B), consistent with literature assignments (39). The multibond correlation from the uracil H-6 to C-1' was also detected (not shown), thus verifying the linkage of the base group to the ribose. The most salient feature in the sugar region is the detection of the multibond correlations from both H-5'' protons to the 54.6-ppm carbon resonance, thus further validating C-4'' as the nitrogen-substituted carbon (Fig. 6). The remaining multibond correlations in the sugar region verify the assignments of the arabinose and ribose carbon resonances. The 1H and 13C NMR assignments are summarized in Table I.
Comparison of the 13C NMR data for the ArnB product with the starting substrate, UDP-Ara4O (22) (Table I), reveals that the uracil and ribose carbons of both compounds resonate at very similar positions, consistent with the 1H NMR evidence that the enzymatic reaction did not alter the uridine moiety. The 13C NMR data dramatically show that the enzyme-catalyzed amino group substitution is localized to C-4'', which shifts upfield by 40.6 ppm, from 95.2 ppm for UDP-Ara4O in D2O (corresponding to the hydrated form of UDP-Ara4O) (22) to 54.6 ppm for UDP-L-Ara4N in D2O. The C-3'' and C-5'' resonances show much smaller upfield chemical shifts of 6.6 and 5.6 ppm, respectively. The chemical shift change decreases by another factor of 2 at C-2'' to 3 ppm and by a further factor of 10 at C-1'' to 0.3 ppm.
ArnB-catalyzed Transamination of UDP-Ara4OPurified ArnB-C6H in the presence of various amine donors at 7.5 mM preferentially utilizes L-glutamate for the synthesis of UDP-L-Ara4N from 150 µM UDP-Ara4O. The rate of transamination with L-methionine, L-glutamine, and L-alanine is measurable at 5, 2, and 1%, respectively, of the rate observed with L-glutamate (data not shown). No activity was seen with L isomers of asparagine, aspartate, glycine, lysine, ornithine, phenylalanine, or tryptophan. However, the analog L-aminoadipic acid (25) supported the transamination of UDP-Ara4O at about 50% the rate of L-glutamate (not shown).
As shown in Fig. 7, the extent of conversion of 100 µM UDP-Ara4O to UDP-L-Ara4N increases as the molar ratio of amine donor to UDP-Ara4O is increased. At a 10-fold excess of L-glutamate, only 53% of the UDP-Ara4O is converted to product, whereas 87 and 91% of the UDP-Ara4O are converted to UDP-L-Ara4N with a 50- or 100-fold excess of L-glutamate, respectively. These findings suggest that that the transamination of UDP-Ara4O is reversible and thermodynamically unfavorable. The equilibrium constant in the direction of UDP-L-Ara4N formation is 0.1.
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The reversibility of the reaction can be demonstrated directly (Fig. 8). In the presence of a 10- or 100-fold excess of -ketoglutarate, 100 µM UDP-L-Ara4N is largely converted back to UDP-Ara4O (Fig. 8, panel A). No UDP-Ara4O is observed in the absence of
-ketoglutarate. The full time course of the reverse reaction with 1 mM
-ketoglutarate is shown in Fig. 8, panel B. The extent of conversion to UDP-Ara4O is consistent with the unfavorable equilibrium constant noted above.
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ArnB Contains a Bound Pyridoxal Phosphate CofactorPyridoxal phosphate-dependent enzymes typically have signature absorbance maxima around 430 and/or 335 nm, depending on whether the pyridoxal 5'-phosphate or the pyridoxamine 5'-phosphate form of the cofactor is predominant (40, 41). The absorbance spectrum of purified ArnB suggests that the cofactor is mostly in the pyridoxal phosphate form, presumably linked as an aldimine to an active site lysine (maximum at
430 nm, Fig. 9) (40). After a 60-min incubation in the presence of excess glutamate, however, the absorbance at
430 nm was greatly decreased, and a concomitant increase in the absorbance at
335 nm was observed (Fig. 9). This is consistent with the conversion of the pyridoxal 5'-phosphate form of the enzyme to the pyridoxamine 5'-phosphate form (40). The results are in accord with the bioinformatic prediction that ArnB is a member of a large family of aminotransferases.
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The presence of the cofactor in the pure protein, albeit mainly in its pyridoxamine 5'-phosphate form, was recently confirmed by x-ray crystallography (25). The authors suggest that use of methionine in some of the purification buffers may account for the fact that the pyridoxal 5'-phosphate form of the cofactor was not predominant in these structural studies.
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DISCUSSION |
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We have now developed the first quantitative assay for ArnB and demonstrated that the purified E. coli enzyme is indeed a pyridoxal-dependent aminotransferase that generates UDP-L-Ara4N from UDP-Ara4O in the presence of glutamate (Fig. 2, box). E. coli ArnB, either with or without a hexa-histidine tag, was expressed using an IPTG-inducible T7lac promoter construct in E. coli NovaBlue(DE3), a polymyxin-sensitive host. In each case, the expression of a 43-kDa protein (Fig. 3) correlated with the overproduction of L-glutamate-dependent conversion of UDP-[14C]Ara4O to the faster migrating nucleotide, presumed to be UDP-L-[14C]Ara4N; both the native and the hexa-histidine tagged proteins displayed similar specific activities (data not shown). Both of ArnB hexa-histidine fusion proteins were readily purified by standard nickel-chelation chromatography (Fig. 3), yielding about 8 mg of pure protein per liter from induced cells with a specific activity of about 1300 nmol/min/mg under standard assay conditions in the forward direction.
By using purified ArnB, we prepared 1.7 mg of the putative UDP-L-Ara4N product. NMR spectroscopy (Figs. 4,5,6) demonstrated unambiguously that the material synthesized by ArnB from UDP-Ara4O is indeed UDP-L-Ara4N. In particular, the location of the amine group at position 4'' and the stereochemistry of the pyranose moiety, as judged by the analysis of the coupling constants and chemical shifts (Table I), are essentially the same as those in the L-Ara4N moiety attached to lipid A (15, 36). To our knowledge, UDP-L-Ara4N is the first example of a nucleoside diphosphate 4''-amino pentose, featuring a pyranose ring. The only reported 4''-amino hexose nucleoside diphosphate is GDP-4-amino-4,6-dideoxymannose (GDP-perosamine), which is a precursor of lipopolysaccharide O-antigens in some Gram-negative bacteria (43). Several 3''-amino hexose nucleoside diphosphates have also been reported; these are precursors of the sugar moieties present in some macrolides and glycopeptide antibiotics (44, 45).
Close orthologs of ArnB are found in all Gram-negative bacteria known to modify their lipid A with L-Ara4N (24). These include, in addition to E. coli, all strains of Salmonella, Y. pestis, P. aeruginosa, and B. cepacia. Additionally, ArnB displays somewhat lower but still very significant sequence homology to the above-mentioned aminotransferases involved in the synthesis of the perosamine present in lipopolysaccharide O-antigens, or the amino sugars found in some macrolide and glycopeptide antibiotics (45). Like ArnB, each of these aminotransferases displays a strong preference for L-Glu as the amine donor (45). Multiple sequence alignments indicate that ArnB contains a highly conserved lysine residue (188), which presumably forms a Schiff's base with the pyridoxal phosphate cofactor that is detected in the ultraviolet visible absorption spectrum at 430 nm (Fig. 9). The recently reported crystal structure of 3-amino-5-hydroxybenzoic acid synthase likewise shows the cofactor covalently bound to the analogous Lys (188) through the expected internal aldimine linkage (46). Our spectroscopic data (Fig. 9) are consistent with ArnB being isolated mainly in the pyridoxal phosphate (aldimine) form of the cofactor, presumably linked to Lys-188. In the presence of excess L-Glu (Fig. 9), the bound pyridoxal phosphate cofactor is converted to the pyridoxamine phosphate derivative. The latter would be used directly to transfer the amine group to UDP-Ara4O (Fig. 2).
The UDP-L-Ara4N (Figs. 4,5,6) generated by ArnB in vitro provides a new tool with which to explore the later steps of the pathway for the modification of lipid A with L-Ara4N (Fig. 2). In cell extracts derived from polymyxin-resistant E. coli, UDP-L-Ara4N can be metabolized to what appears to be a 4''-formyl-amino derivative (22). A separate catalytic domain of ArnA catalyzes formyl group transfer from N-10-formyltetrahydrofolate to the 4''-amine of UDP-L-Ara4N to make the proposed nucleotide UDP-Ara4-formyl-N (Fig. 2).3 The role of this formylated nucleotide in the modification of lipid A with L-Ara4N is not immediately obvious, given that undecaprenyl phosphate--L-Ara4N (16, 17) is the Ara4N donor used by ArnT for transferring the L-Ara4N moiety to lipid A.
Biosynthesis of the UDP-L-Ara4-formyl-N intermediate in the cytoplasm (Fig. 2) might compensate for the relatively unfavorable equilibrium constant for the conversion of UDP-Ara4O to UDP-L-Ara4N, which we estimate to be about 0.1 (Figs. 7 and 8). Preliminary results suggest that ArnC might actually be selective for UDP-L-Ara4-formyl-N, as proposed in Fig. 2.3 However, deformylation would then be necessary prior to L-Ara4N transfer to lipid A from undecaprenyl phosphate--L-Ara4N (16, 17). It would make sense for the proposed deformylation to occur in the periplasm in order to avoid a futile cycle in the cytoplasm (Fig. 2). Furthermore, if the putative inner membrane transporter (flippase) for undecaprenyl phosphate-
-L-Ara4-formyl-N (Fig. 2) were unable to transport undecaprenyl phosphate-
-L-Ara4N, accumulation of the latter on the outer surface of the inner membrane would take place, thereby ensuring that ArnT has an adequate supply of its donor substrate with which to modify lipid A prior to its export to the outer membrane. The latter process occurs very rapidly (within a minute) in wild-type cells (47). The biochemical characterization of the formyltransferase activity of ArnA (22), together with the development of assays for ArnC and the putative deformylase (Fig. 2), should reveal fundamental new insights into the compartmentalization of lipid A modification enzymes and the mechanisms of lipid transport across the inner membrane.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.
1 The abbreviations used are: LPS, lipopolysaccharide; L-Ara4N, 4-amino-4-deoxy-L-arabinose; UDP-L-Ara4N, uridine-5'-diphospho--(4-amino-4-deoxy-L-arabinose); UDP-Ara4O, uridine 5'-(
-L-threo-pentapyranosyl-4''-ulose diphosphate); UDP-GlcUA, UDP-glucuronic acid; pEtN, phosphoethanolamine; IPTG, isopropyl-1-thio-
-D-galactopyranoside; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)-methane; Kdo, 3-deoxy-D-manno-2-octulosonic acid; NOE, nuclear Overhauser effect.
2 The 1H chemical shifts given in the text and in Table I refer to the experiment shown in Fig. 4. The actual chemical shifts in some of the other experiments (such as the COSY analysis in Fig. 5) may vary slightly (by 0.02 ppm). The analysis in Fig. 5 was done several weeks after that shown in Fig 4, possibly allowing for small changes in the volume or pD of the sample.
3 S. D. Breazeale and C. R. H. Raetz, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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