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J. Biol. Chem., Vol. 281, Issue 16, 10778-10785, April 21, 2006
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From the
State Key Laboratory of Microbial Resources at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China, the
Institut für Genomforschung, Universität Bielefeld, 33615 Bielefeld, Germany, and the ¶Graduate School of Chinese Academy of Sciences, Beijing 100039, People's Republic of China
Received for publication, December 12, 2005 , and in revised form, January 23, 2006.
| ABSTRACT |
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| INTRODUCTION |
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-D-glucopyranoiside, is the major low-molecular mass thiol in mycobacteria and streptomycetes (2). Some investigations showed that mycothiol is associated with protection of Mycobacterium tuberculosis and Mycobacterium smegmatis against antibiotics such as rifampin (3, 4) and also helped these pathogens to detoxify reactive oxygen species produced by host cells (5). Thus, mycothiol is a potential target for medical treatment and has raised interest from both academia and industry. In addition, mycothiol also has the ability to protect cells against a range of toxic compounds. For example, in Amycolatopsis methanolica and Rhodococcus erythropolis, mycothiol detoxifies formaldehyde by acting as a cofactor for a formaldehyde dehydrogenase (6) and detoxifies alkylating agents such as monobromobimane by converting them to S-conjugates of mycothiol (7, 8). To date, the understanding of the physiological function of mycothiol is limited to detoxification and the protection of living cells (9), whereas essential metabolic roles for cell growth have not been reported. A survey on the distribution of low-molecular mass thiols in microorganisms showed that Corynebacterium diphtheriae produced mycothiol (2). However, the occurrence of mycothiol in other Corynebacterium species has not been reported, and the physiological function of mycothiol in corynebacteria is still not well defined. The genus Corynebacterium covers both medical and industrial important species. For example, Corynebacterium glutamicum is commercially used for production of amino acids and vitamins (10), and C. diphtheriae is a pathogen for human beings. Recently, the genomes of C. glutamicum (11, 12), Corynebacterium efficiens, C. diphtheriae (13), and Corynebacterium jeikeium (14) have been sequenced. This greatly stimulated the study of the physiology of corynebacteria. Consequently, C. glutamicum has been newly characterized for its robust ability to metabolize aromatic compounds (1517), and a novel glutathione-independent gentisate pathway has been described (18).
The biosynthesis of mycothiol was characterized in M. smegmatis and M. tuberculosis, and the genes mshB, mshC, and mshD were found encoding for the enzymes that sequentially catalyze the formation of mycothiol from 1D-myo-inosityl-2-acetamido-2-deoxy-
-D-glucopyranoside (GlcNAc-Ins). A fourth gene, mshA, is involved in the production of GlcAc-Ins, but the substrate of MshA has not been identified (9). Orthologs of these msh genes could be found in the genome data of the Corynebacterium species, but their functions in biosynthesis of mycothiol have not been proven experimentally.
During our studies on biosynthesis of mycothiol and assimilation of aromatic compounds with C. glutamicum, we found that mycothiol-negative mutants lost the ability to grow on several aromatic compounds. This surprising discovery raised the question of how mycothiol is involved in aromatic compound assimilation/degradation. In this report, our results indicate that mycothiol functions as an essential growth factor for C. glutamicum when gentisate and 3-hydroxybenzoate are provided as carbon sources. We further linked the biosynthesis of mycothiol to gentisate assimilation by identification of a mycothiol-dependent maleylpyruvate isomerase in C. glutamicum.
| EXPERIMENTAL PROCEDURES |
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ncgl1055, pK18mobsacB
ncgl1457, and pK18mobsacB-
ncgl2487 were constructed using the gene-SOEing method described by Horton (19). The primers used are listed in Table 1. The primary products were amplified using fusion DNA polymerase (New England Biolabs). The resulting products were purified using the PCR purification kit (Qiagen, Hilden, Germany) and then used as templates for the second round of PCR. The final products were digested with restriction enzymes corresponding to the cleavage sites introduced via PCR and ligated into appropriately digested pK18mobsacB. The ligation mixture was used to transform E. coli DH5
MCR, the transformants were selected on LB plates containing 50 µg ml1 kanamycin and 40 µg ml1 X-Gal (5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside).
To generate mutants in mycothiol biosynthesis from C. glutamicum, site-specific gene deletion was performed using the above generated pK18mobsacB derivatives that are not replicable in C. glutamicum and allow for marker-free deletion of the target genes (20). The resulting plasmids pK18mobsacB
ncgl1055, pK18mobsacB
ncgl1457, and pK18mobsacB
ncgl2487 were transformed into C. glutamicum ATCC 13032 by electroporation (21). Integration of the introduced plasmids into the chromosome by single crossover was tested by selection on BHIS plates containing 25 µgml1 kanamycin. For the deletion of the target gene, the kanamycin-resistant (KmR) cells were grown overnight in liquid BHIS and spread on BHIS plates containing 10% sucrose. Cells growing on this plate were tested for kanamycin sensitivity (KmS) by parallel picking on BHIS plates containing either kanamycin or sucrose. Sucrose-resistant and kanamycin-sensitive cells were then tested for deletion by PCR, using the corresponding DF1 and DR2 primer pair (Table 1).
Extraction and Determination of Mycothiol in Wild Type and Mutants of C. glutamicumThe detection of mycothiol from C. glutamicum cells was carried out according to the procedures as described by Newton et al. (2) and was modified. One milliliter of 50% warm (60 °C) acetonitrile containing 20 mM Tris-HCl, pH 8.0, and 2 mM bromobimane (Sigma) was added to a tube that contained 200 mg of frozen cells, sonificated at 60 °C for 20 s, and maintained in a 60 °C water bath for 15 min in dark. After acidification with 5 µl of 5 N methanesulfonic acid, the cellular debris was removed by centrifugation at 10,000 x g for 10 min. Supernatants were diluted 3-fold in aqueous 10 mM methanesulfonic acid prior to HPLC3 analysis. The bimane derivatives of various thiols were separated and detected with HPLC that was equipped with a C18 column (ZORBAX, 250 x 4.6 mm) and was operated at the following conditions. The column was first eluted with 10% methanol (in water) for 5 min, and then the methanol content was increased to 100% in 10 min. The bimane derivative of mycothiol was eluated at 11.79 min in this system.
Heterologous Expression of the Maleylpyruvate Isomerase Gene in E. coli and Purification of Recombinant ProteinThe expression plasmid pET28a-ncgl2918 was described previously (18). The plasmid was electroporated into E. coli BL21(DE3). Synthesis of recombinant protein in E. coli BL21(DE3) cells was initiated by addition of 0.5 mM isopropyl 1-thio-
-D-galactopyranoside when the culture reached A600 of 0.60.8 and continued cultivation for an additional 8 h at 25°C. Cells were harvested by centrifugation and were disrupted by sonification at 4 °C (160 W, 3 s sonifying versus 5 s break, 50 cycles) in Tris-HCl buffer (20 mM Tris-HCl, pH 8.0). Cellular lysate was centrifuged and the supernatant was used for protein purification. Recombinant protein was purified with the His-Bind protein purification kit (Novagen, Madison, WI) according to the manufacturer's instructions. To remove any residue proteins, imidazole, and salts in the collected fractions, fractions were pooled and were further separated by SuperdexTM 200 gel chromatography with Tris-HCl buffer (20 mM Tris-HCl, pH 8.0). All steps of chromatography were controlled by the fast protein liquid chromatography system (Äkta FPLC, Amersham Biosciences). The purified protein was concentrated by ultrafiltration through Millipore Ultra-15 (10 kDa) and stored at 70 °C.
Determination of Molecular Mass of the Purified ProteinsThe native molecular mass of the maleylpyruvate isomerase was estimated by gel filtration chromatography on a prepacked Superdex 200 column (Amersham Biosciences). The column was equilibrated and eluted with 50 mM Tris-HCl (pH 7.6) containing 100 mM NaCl at a flow rate of 0.4 ml min1. Molecular mass was calculated according to their elution volume and calibrated with the molecular mass standard kit (MW-GF-1000, Sigma). The subunit molecular mass was determined with SDS-PAGE, which was conducted with a 5% stacking gel and 12% resolving gel and run in a Mini-PROTEIN II Electrophoresis Cell (Bio-Rad) according to the manufacturer's instructions. Apparent molecular mass was estimated according to the relative mobility to protein standards with molecular mass ranging from 14 to 97 kDa.
Enzymatic Preparation of Maleylpyruvate and Determination of Maleylpyruvate Isomerase ActivityMaleylpyruvate was prepared from gentisate by gentisate 1,2-dioxygenase digestion (18) in a system containing (total volume 3 ml): 0.1 mM gentisate, 5 µl of recombinant 1,2-dioxygenase from E. coli, 50 mM Tris-HCl buffer, pH 8.0. After incubation at room temperature for 3 min (complete conversion of gentisate to maleylpyruvate), this mixture was used as the crude maleylpyruvate preparation without any further purification. For determination of maleylpyruvate isomerase activity, 10 µl of boiled cellular lysate of C. glutamicum and 510 µl of maleylpyruvate isomerase preparations were added to the above crude maleylpyruvate preparation. The maleylpyruvate isomerase activity was qualitatively monitored by scanning the spectral absorption changes at 250400 nm with a UV visible spectrophotometer (Beckman Coulter DU800) at a wavelength interval of 1 nm, and was quantitatively determined by measuring the decrease of absorbance at 330 nm due to maleylpyruvate disappearance. Rates of isomerization of maleylpyruvate to fumarylpyruvate were calculated with a value of 2,400 cm1 M1 for the extinction changes at 330 nm (22). To test if coenzyme A and cysteine support maleylpyruvate isomerase activity, coenzyme A and cysteine (each 0.01 mM) were included in the assay broth. Protein concentration was determined according to the method of Bradford (23), with bovine serum albumin as the standard.
Preparation and Treatment of Various Bacterial Cellular LysatesTo evaluateifthefollowingbacteriacontainedthefactorthatsupportedmaleylpyruvate isomerase from C. glutamicum, cells of about 0.15 g from each of S. clavuligerus, B. megaterium, B. subtilis, S. coelicolor, and mutants of mshB, mshC, and mshD of C. glutamicum were disrupted by sonification at 4 °C (160 watts, 3 s sonifying versus 5 s break, 50 cycles) in Tris-HCl buffer (20 mM Tris-HCl, pH 8.0). Cellular debris was separated by centrifugation and the resulting supernatants were boiled for 5 min to eliminate any enzyme activities. The boiled supernatants (10 µl) were added to the maleylpyruvate isomerase activity assay mixtures, as described above.
Isolation of Mycothiol from C. glutamicumIsolation and purification of mycothiol from C. glutamicum was carried out by integration of Sephadex LH20 chromatography and the methods of Newton et al. (24) with a thiol-affinity chromatography and Steenkamp et al. (25) with HPLC. Frozen cells (37 g) of C. glutamicum were thawed in 74 ml of 0.75 M perchloric acid. Acid-insoluble cellular debris was removed by centrifugation at 12,000 x g for 10 min. The supernatant was adjusted to pH 4.5 with 4 M KOH and then stored at 0 °C for 30 min. Precipitated potassium perchlorate was removed by centrifugation at 10,000 x g for 10 min. The supernatant (81 ml) was mixed with 27 ml of buffer A (0.4 M Tris-HCl, 2 M NaCl, 4 mM EDTA, pH 7.5). The mixture was applied onto a thiopropyl-SepharoseTM 6B column (Amersham Biosciences). The column was washed using 80 ml of buffer B (0.1 M Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 7.5). The column was then eluted with 60 ml of buffer C (0.1 M Tris-HCl, 1 mM EDTA, 30 mM dithiothreitol, pH 7.5). Regeneration of this thiolpropyl-Sepharose 6B column was performed with 1.5 mM 2,2'-dipyridyl disulfide, according to instructions from the Thiolpropyl-Sepharose 6B supplier (Amersham Biosciences, catalog number 17-0420-01). The active eluant (2 ml) was applied onto a Sephadex LH20 column (Amersham Biosciences) and fractionated using 50% methanol in water, pH 4.5. The active fractions were concentrated with a rotary evaporator (Ratavapor, BÜCH RE121, Switzerland), lyophilized, and finally dissolved in water. Further purification of mycothiol was carried out with HPLC using isocratic elution (2% acetonitrile, 0.45% propionic acid) on a C18 column (ZORBAX, 250 x 4.6 mm).
Mass SpectrometryPositive-ion electrospray mass spectrometry analyses were performed on an Agilent 1100 LC/MSD Trap XCT instrument (Agilent Technologies, Palo Alto, CA). Instrument control, data acquisition, and data processing were done using the Agilent 1100 Chemstation (version 10.02) and Data Analysis (version 5.1) software. The instrument was operated in full-scan mode. Acquisition was performed in the continuum mode, and the acquisition parameters were as follows: tune source: trape drive (39.9), octopole RF amplitude (187.1 vpp), capillary exit (94.8 V), skim 1 (40.0 V), skim 2 (5.0 V), dry temperature (325 °C), nebulizer (50.00 p.s.i.), nitrogen gas (10.00 liters/min), HV capillary (2680 V), HV end-plate offset (500 V). The scan (average of three spectra) was between m/z 100550 with maximal Accu time of 200,000 µs and ICC target of 200,000. Fragmentation was set with SmartFrag Ampl between 30 and 200%, fragmentation width, 10.00 m/z; fragmentation time, 40,000 µs; and fragmentation delay, 0 µs.
Kinetic MeasurementsMichaelis-Menten kinetics of the maleylpyruvate isomerization catalyzed with isomerase from C. glutamicum was identified by plotting reaction rates against substrate concentrations (ranging from 66 to 380 µM) at a mycothiol concentration of 2.5 µM. The apparent Km and Vmax values were determined by nonlinear regression analysis of the plots of the initial isomerization rates and substrate concentrations.
Effects of Metal Ions and Chemicals on Maleylpyruvate Isomerase ActivityTo test the effects of various metal ions and chemicals on the mycothiol-dependent maleylpyruvate isomerase, Mg2+, Ca2+, Cu2+, Ni2+, Mn2+, Fe3+, Zn2+ (each 1 mM), GSH (0.05 mM), dithiothreitol (0.05 mM), EDTA (1 and 10 mM), and bromobimane (1 mM) were incubated in 20 mM Tris-HCl, pH 8.0, with the enzyme for 15 min at room temperature. The activities of the treated enzymes were assayed as described above. The enzyme without treatment was run in parallel as control.
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| RESULTS |
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-ketoadipate pathway (15), (iii) resorcinol was assimilated through the hydroxyquinol 1,2-dioxygenase pathway (16), and (iv) 3-hydroxybenzoate as well as gentisate were assimilated through a glutathione-independent gentisate pathway (18). Thus, we deduced that there might be a link between the glutathione-independent gentisate pathway and mycothiol biosynthesis in C. glutamicum.
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This mycothiol-dependent maleylpyruvate isomerase from recombinant E. coli was analyzed by SDS-PAGE and gel filtration chromatography. Results showed a single protein band at 33 kDa in the SDS-PAGE and a molecular mass of 34 kDa in gel filtration chromatography. Thus, this mycothiol-dependent maleylpyruvate isomerase of C. glutamicum is a monomeric enzyme. Amino acid sequence analysis revealed a cysteine residue at position 61 (Cys61). Treatment of the enzyme with thiolmodifying agents such as bromobimane, N-ethylmaleimide, p-chloromercuribenzoate, and iodoacetamide did not significantly inhibit the activity of the enzyme (18), indicating that the cysteine residue in this protein is not important for enzyme activity. Furthermore, alignment of several Ncgl2918 orthologs showed that the cysteine residue is not conserved (Fig. 3). In addition, site-directed mutation of this cysteine residue into the alanine residue did not cause a loss in enzymatic activity (data not shown). Divalent metal ions including Mg2+, Ca2+, Cu2+, Ni2+, and Mn2+ (each 1 mM) did not affect the activity of the mycothiol-dependent maleylpyruvate isomerase. Fe3+ (1 mM) showed a moderate inhibition with a residual activity of 85%. Zn2+ (1 mM) showed a moderate stimulation of activity to 121%. EDTA at 1 mM slightly increased activity, but when its concentration reached 10 mM, the activity was inhibited completely. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 ± 11.9 µM and 1520 ± 57.4 µmol/min/mg, respectively.
| DISCUSSION |
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Mycothiol plays important roles in detoxification of thiol-reactive substances such as formaldehyde and some antibiotics (3, 5, 29). C. glutamicum was originally isolated from soil, and its strong ability to assimilate aromatic compounds has been described recently (16). The finding that mshC and mshD mutants of C. glutamicum have lost the ability to grow on gentisate and on 3-hydroxybenzoate was surprising, because no links between mycothiol biosynthesis and aromatic compound assimilation had been established. By examination of all enzymatic reactions involved in the pathway for gentisate and 3-hydroxybutyrate assimilation, we have proven that the isomerization of maleylpyruvate into fumarylpyruvate needs mycothiol as cofactor and that this is the key step that couples the mycothiol biosynthesis and the gentisate/3-hydroxybenzoate assimilation processes. In a very comprehensive review on the mycothiol biochemistry, Newton and Fahey wrote that "there may also be mycothiol-dependent processes, but their discovery will likely be more difficult" (9). To the best of our knowledge, this is the first time that mycothiol biosynthesis was found to be essential for cell growth when aromatic compounds are provided as carbon sources, and that a mycothiol-dependent maleylpyruvate isomerase was identified.
Gentisate and substituted gentisates are key intermediates during aerobic degradation of many aromatic compounds, such as 3-hydroxybenzoate (26, 33, 34), 3,5- or 2,5-xylenol (35, 36), salicylate (3739), 3,6-dichloro-2-methoxybenzoate (40), and naphthalene (4143). In the gentisate pathway, isomerization of maleylpyruvate to fumarylpyruvate is catalyzed by either a GSH-dependent maleylpyruvate isomerase (26, 27, 34), a mycothiol-dependent maleylpyruvate isomerase (this study), or a maleylpyruvate isomerase as in B. megaterium that does not rely on glutathione or mycothiol (22). The discovery of a mycothiol-dependent maleylpyruvate isomerase in C. glutamicum introduces a new category of maleylpyruvate isomerase, and constitutes significant progress on the way to understanding the gentisate pathway of aromatic assimilation/degradation. Considering the many similarities between mycothiol and glutathione metabolism, we believe that the mycothiol-dependent maleylpyruvate isomerase is only one representative of enzymes that are analogous to glutathione-dependent glyxoylases, S-transferases, thioltransferases, etc., and that more mycothiol-dependent enzymes will be identified in the near future.
| FOOTNOTES |
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1 Both authors contributed equally to this work. ![]()
2 To whom correspondence should be addressed: Institute of Microbiology, Chinese Academy of Sciences, Zhong-Guan-Cun, Haidian, Beijing 100080, P. R. China. Tel.: 86-10-62527118; Fax: 86-10-62652317; E-mail: liusj{at}sun.im.ac.cn.
3 The abbreviation used is: HPLC, high performance liquid chromatography. ![]()
| REFERENCES |
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