Mycobacterial Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
Received on April 9, 2002; revised on August 6, 2002; accepted on August 7, 2002
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Abstract |
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Key words: glyceraldehyde/isopentenyl diphosphate synthesis/pyruvate/transketolase
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Introduction |
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In human tissues IPP is synthesized through the acetate/mevalonate pathway, and this was assumed to be true for bacteria as well. However, a series of studies (Duvold et al., 1997; Horbach et al., 1993
; Lois et al., 1998
; Putra et al., 1998a
,b; Rohmer et al., 1993
; Sprenger et al., 1997
; Zhou and White, 1991
) have clearly shown that some bacteria, including Mycobacterium phlei (Putra et al., 1998a
), use an entirely different pathway for IPP biosynthesis. Through in vivo feeding experiments utilizing Escherichia coli (Rohmer et al., 1993
), it was demonstrated that the mevalonate-independent pathway utilizes pyruvate and D-glyceraldehyde 3-phosphate (GAP) to form isoprenoid compounds. The first step in this synthesis is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP; Figure 1). The enzyme responsible for catalyzing this reaction in E. coli is 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Once DXP has been synthesized a number of other biosynthetic steps are required for the synthesis of IPP (Rohdich et al., 2001
).
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Results |
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BLAST searches
BLAST searches of the M. tuberculosis genome also supported the hypothesis that M. tuberculosis utilized the mevalonate-independent pathway for IPP synthesis. The searches did not reveal any genes that encode proteins with significant homology to known enzymes, such as 3-hydroxy 3-methylglutaryl coenzyme A reductase or mevalonate kinase of the mevalonate pathway for IPP biosynthesis. The searches did identify a number of genes that encode proteins with homology to previously identified enzymes in the nonmevalonate pathway. Two of these genes, Rv2682c and Rv3379c, encode proteins that are similar in primary structure to the DXS of E. coli (38% and 33% identity, respectively) and have a high degree of homology to each other (48.9% identity, Figure 2).
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Activity of the recombinant protein was determined in varying concentrations of GAP and the enantiomers of GA and D-erythrose 4-phosphate in the presence of saturating levels of pyruvate (Figure 5). The activity of the recombinant protein was also determined in varying concentrations of pyruvate in the presence of saturating levels of DL-glyceraldehyde. The Kmpyruvate was determined to be 40 µM. The KmGAP was three orders of magnitude lower than either the KmD-glyceraldehde or the KmL-glyceraldehyde at 6.1 µM (Figure 5). Rv2682c presumably utilizes the L-glyceraldehyde to form 1-deoxy-L-ribulose, although the exact nature of the product seen by TLC was not determined. The enzyme was also able to utilize D-erythrose 4-phosphate as a substrate with a Km of 121 µM (Figure 5). In this case the product is likely 1-deoxy-D-fructose 6-phosphate. Rv2682c was not able to utilize either D-ribose 5-phosphate or -ketobutyric acid as substrates.
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Discussion |
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Interestingly, the M. tuberculosis genome contains two genes Rv2682c and Rv3379c that have significant homology at the amino acid level to E. coli DXS and to each other. Thus far only two other organisms have been reported to have two homologs of DXS: the photosynthetic Rhodobacter capsulatus (Hahn et al., 2001) and the soil bacterium Streptomyces coelicolor (Cane et al., 2001
). In both cases both proteins were functional. The most obvious differences between the proteins encoded by the genes in M. tuberculosis are two apparent deletions (Figure 2). The N-terminus of Rv3379c appears to have a 65-amino-acid truncation. Inspection of the genome reveals that Rv3379c may have been interrupted by an adjacent insertion element (IS6110), which would account for the truncation of the protein. In addition, the amino acids corresponding to positions 193228 of Rv2682c are not found in Rv3379c.
The M. tuberculosis DXS homologs have a high degree of similarity to previously identified DXS and other transketolase enzymes. The genes both encode the conserved DRAG sequence that has been proposed as a DXS signature sequence (Hahn et al., 2001). Rv2682c has all 13 conserved amino acid residues that have been identified as being important in the function of transketolase enzymes (Hahn et al., 2001
), and Rv3379c has 12 of the 13 (Figure 2). However, Rv3379c is missing a critical histidine residue that corresponds to His49 of the E. coli enzyme. His49 of the E. coli DXS enzyme has been shown to be essential for catalytic activity (Querol et al., 2001b
). This residue aligns with a conserved histidine residue in yeast transketolase (His30) that has also been shown to be involved in catalysis (Wikner et al., 1997
). These observations strongly suggest that Rv3379c does not encode a functional DXS enzyme. This hypothesis is supported by the observation that when the gene was cloned and expressed (data not shown) under conditions similar to those used for Rv2682c the soluble protein is inactive in standard DXS activity assays. However, this is a negative result and, as such, can only be accepted with equivocation.
Although DXP synthases have been cloned from several sources, including E. coli (Kuzuyama et al., 2000; Lois et al., 1998
; Sprenger et al., 1997
), Streptomyces spp. (Cane et al., 2001
; Kuzuyama et al., 2000
), Rhodobacter capsulatus (Hahn et al., 2001
), Pseudomonas aeruginosa (Altincicek et al., 2000
), Synechococcus leopoliensis (Miller et al., 1999
), and several plant species (Lange et al., 1998
; Bouvier et al., 1998
; Estevez et al., 2000
), and there is currently considerable interest in screening for inhibitors of DXS activity as potential antibiotics, herbicides, or antimalarials, little information about the enzymatic properties of these transketolases from pathogens has been published. In general, transketolases from yeast, plants and bacteria can utilize a broad range of substrates, whereas those from mammalian sources are more specific (Schenk et al., 1998
). Rv2682c is typical of bacterial transketolases: it acts on a range of substrates and it has kinetic similarities to the other bacterial DXS enzymes for which kinetic data has been reported. However, it also has unique properties. Rv2682c is capable of utilizing D-glyceraldehyde 3-phosphate and D-erythrose 4-phosphate as well as both D- and L-glyceraldehyde as aldose substrates but is not able to utilize D-ribose 5-phosphate. Rv2682c has a Kmpyruvate similar to the values of 96 µM and 65 µM reported for the enzymes from E. coli and Streptomyces sp. strain CL190 (Kuzuyama et al., 2000
) but is considerably lower than the values between 300 µM and 3 mM reported for the enzymes from R. capsulatus (Hahn et al., 2001
) and S. coelicolor (Cane et al., 2001
). Rv2682c has a KmGAP that is 20- to 100-fold lower that the Km values reported for DXS enzymes from other bacteria (Cane et al., 2001
; Hahn et al., 2001
; Kuzuyama et al., 2000
). This observation is even more notable when one considers that only about 4% of the GAP is in the aldehyde form in solution (Swenson and Barker, 1971
). The KmD-glyceraldehyde calculated for Rv2682c is similar to the range (1038 mM) reported for other bacteria (Hahn et al., 2001
; Kuzuyama et al., 2000
). Interestingly the enzyme encoded by Rv2682c is also capable of utilizing L-glyceradehyde and D-glyceraldehyde. The ability to utilize L-glyceraldehyde is unusual, because most transketolases are specific for aldoses with a D-configuration at the C2 atom next to the aldehyde group (Sprenger et al., 1995
).
The cofactors Mg2+ and TPP can be removed from the native DXS activity and can be reconstituted by addition of exogenous cofactors. However, the purified recombinant could not be reconstituted after removal of either of the cofactors, a property that is most likely due to destabilization of the tertiary structure of the enzyme. The KdTPP for the native M. tuberculosis activity is 1 µM in the presence of Mg2+, which is the same as the value reported for yeast transketolase (Heinrich et al., 1972).
The majority of the DXS assays reported in the literature utilize either TLC or high-performance liquid chromatography (HPLC) to quantitate the DXS reaction product (Cane et al., 2001; Estevez et al., 2000
; Kuzuyama et al., 2000
; Lange et al., 1998
; Lois et al., 1998
; Sprenger et al., 1997
). Both of these methods are time- and labor-intensive and thus have limited usefulness in large-scale screening for inhibitors. A fluorometric assay has been devised for the determination of DXS activity for screening inhibitors with potential antibiotic, herbicidal or antimalarial activity (Querol et al., 2001a
). However, the authors report a Kmpyruvate of 2.9 mM for the recombinant E. coli DXS, a value 30-fold higher than that reported for recombinant DXS from the same source using an HPLC-based method (Kuzuyama et al., 2000
). A coupled spectrophotometric assay has also been reported for rapid screening of DXS activity (Altincicek et al., 2000
); the radiometric, ion exchange assay method used here is also amenable to high through-put screening for inhibitors and has the advantage of containing a single enzyme in the reaction mixture.
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Materials and methods |
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PCR amplification and cloning of Rv2682c from M. tuberculosis H37Rv genome
On the basis of the nucleotide sequence of Rv2682c (Cole et al., 1998) the gene was amplified using the following oligonucleotide primers: 5'-ACTAGCCATATGCTGCAACAGATCCG-3' and 5'-ATGAAGCTTCTTAGTCGAGATGTTCTGGAA-3' containing a Hind III and Nde I restriction site (underscored). Polymerase chain reaction (PCR) using a Perkin Elmer GeneAmp 2400 and rTth polymerase (PE Biosystems, Foster City, CA) resulted in the amplification of a single DNA fragment of the expected size (1.6 kb). The PCR fragment was purified from agarose gel after electrophoresis, digested with Nde I and Hind III, and ligated into pET28a+ vector (Novagen, Madison, WI) that had been similarly digested. Plasmids were propagated in E. coli DH5
cells (Life Technologies, Rockville, MD) and were isolated using Qiagen plasmid kits. General molecular biology techniques were done as described earlier (Sambrook et al., 1989
).
Expression and purification of the recombinant Rv2682c
E. coli strain ER2566 (New England Biolabs, Beverly, MA) was used as the expression host. Bacteria harboring the plasmid were grown at 37°C in Luria Bertani broth containing kanamycin (50 µg/ml) to an OD600 ~ 0.6 with shaking. The culture was allowed to cool on ice, and ispropylthiogalactoside was added to a final concentration of 50 µM, and the culture was incubated overnight at 15°C with shaking. Cells were harvested by centrifugation at 8000 x g for 10 min and stored at 80°C. The frozen cells were resuspended in a lysis buffer (2 ml/g) containing 50 mM 4-morpholine propane sulfonic acid (MOPS) (pH 7.9), 10 mM MgCl2, 15% glycerol, and 1 mM 2-mercaptoethanol, broken by sonication on ice and then centrifuged for 40 min at 20,000 x g. The volume of the supernatant was adjusted to 30 ml, and imidazole was added to achieve a final concentration of 5 mM. A packed volume of 0.75 ml of Talon metal affinity resin (Clontech, Palo Alto, CA) pre-equilibrated with lysis buffer was added to the resulting solution and rocked at 4°C for 20 min. A small column was packed with the slurry, and the column was washed sequentially with 2 ml lysis buffer followed by 45 ml lysis buffer containing 7 mM imidazole and 750 mM NaCl. The His-tagged Rv2682c was eluted with 2-ml volumes of lysis buffer containing 25 mM, 50 mM, 100 mM, and 200 mM imidazole, and each fraction was analyzed by SDSPAGE. Although some recombinant protein eluted in all fractions, the majority was found in the 100 mM imidazole fraction. The 100 and 200 mM imidazole fractions were pooled concentrated and desalted on a PD-10 desalting column (Amersham, Piscataway, NJ). Recombinant Rv2682c was then concentrated by ultrafiltration using a 5000 molecular weight cutoff Centricon (Millipore, Bedford, MA). Glycerol was added to a final concentration of 20% and the resulting mixture was divided into aliquots and stored at 80°C.
Partial purification of native DXS
M. tuberculosis (H37Rv) was grown to mid-log phase in glycerol-alanine-salts medium, harvested by centrifugation and washed with saline. The resulting pellet was irradiated for 18 h at 2315 Rads/min using a JL Shepard instrument with a 137Cs source. This exposure was calculated to kill 100% of the bacteria but retain 90% of enzyme activity. Irradiated H37Rv cells were suspended in ice-cold homogenization buffer containing 50 mM MOPS (pH 7.9), 0.25 M sucrose, 10 mM MgCl2, and 5 mM 2-mercaptoethanol and subjected to probe sonication on ice for 10 cycles of 60 s on and 90 s off in a Soniprep 150 (Integrated Services, TCP, Palisades Park, NJ). All steps were done at 4°C. The homogenate was centrifuged at 21,000 x g for 15 min, and the resulting pellet (debris and cell wall) was discarded. The supernatant was then spun at 200,000 x g in a Beckman 70.1 Ti rotor for 1 h. To remove a competing enzymatic activity that interfered with the DXS assay the 200,000 x g supernatant (cytosol) was loaded onto a 15-ml DE-52 anion exchange column pre-equilibrated with homogenization buffer. Protein was eluted with a NaCl gradient from 01 M and collected in 1.5-ml fractions. Fractions were assayed for DXS activity as described later, and those containing activity were pooled; this resulted in a twofold increase in the specific activity of DXS and removal of the interfering activity. Glycerol was added to a final concentration of 30%, and the resulting mixture was divided into 100-µl aliquots and frozen at 80°C. Endogenous, tightly bound divalent cations were removed from the partially purified protein by cation exchange on BioRex 70. The anion exchange purification step served to remove bound TPP from the native protein. Attempts to remove divalent cations or TPP from the purified recombinant protein resulted in irreversible loss of enzymatic activity.
In vitro DXS assay using GA
DXS activity was assayed in a mixture containing 100 mM MOPS (pH 7.9), 5 mM TPP, 120 µM [2-14C]pyruvate, 26 mM GA and 1 µg of recombinant or 50100 µg of partially purified protein in a final volume of 75 µl. Assays were incubated at 37°C for 30 min and stopped by the addition of 1 ml 50% methanol. Reaction mixtures where then passed over an anion exchange column (1.5 ml bed volume in a Pasteur pipette) of AG1-X2. The radiolabeled product, DX, was eluted with 5 ml 50% methanol. A 1-ml aliquot of eluate was taken and dried under N2, resuspended in 250 µl 50% methanol, and an aliquot was subjected to liquid scintillation spectrometry. Aliquots were also spotted on TLC plates (silica gel 60) along with authentic DX, and the plates were developed in n-propanol/ethyl acetate/H2O (6:1:3 v/v/v) as previously described (Lois et al., 1998). Radiolabeled product was located with a Bioscan System 200 Imaging System (Bioscan, Washington, DC), and authentic standard DX was located with anisaldehyde spray reagent (Dunphy et al., 1967
).
In vitro DXS assay using GAP
To assay activity in the presence of GAP typical reaction mixtures contained 250 mM MOPS (pH 7.9), 5 mM TPP, 120 µM [2-14C]pyruvate, recombinant or partially purified protein and various concentrations of GAP in a final volume of 75 µl, which were incubated at 37°C for 30 min. In this case reactions were terminated by heating at 80°C. Following centrifugation at 13,000 x g for 5 min, the supernatant was transferred to new tubes and aliquots were counted in a liquid scintillation counter. In addition, aliquots were spotted on silica gel 60 TLC plates along with authentic DXP; radiolabeled enzymatic products were separated from [2-14C]pyruvate using n-propanol/ethyl acetate/H20 (6:1:3) (Lois et al., 1998) and located with the Bioscan imaging system. To confirm the identity of the radiolabeled DXP, some of the radiolabeled enzymatic products were treated with bovine alkaline phosphatase and spotted on silica gel 60 TLC plates along with authentic DX. Standard DXP and DX were located by spraying with an anisaldehyde spray reagent (Dunphy et al., 1967
).
Enzymatic properties of DXS
Determination of optimal pH was done using the GA-based assay essentially as already described. However, a broad pH range buffer (0.05 M Tris/0.025 M MES/0.025 M acetic acid) adjusted with tetraethylammonium hydroxide was used, and activity was determined at 0.5 pH unit intervals between pH 5.5 and 9.0. To determine the Km for pyruvate and GA, concentrations of either the [2-14C]pyruvate and GA were varied while the other was held constant, and the radiolabeled product was assayed as already described. The optimal concentrations of TPP and divalent cation were determined in the same manner; however, partially purified native protein was used.
Other procedures
Protein concentrations were estimated using a BCA protein assay kit (Pierce, Rockford, IL). All enzyme assays were done under conditions that were linear for both time and protein concentration, and the data presented are averages of duplicate assays from representative experiments. Radioactivity was measured in Ecolume (ICN) using a Beckman (Fullerton, CA) LS 6500 liquid scintillation spectrometer. Colorado State University Macromolecular Resources synthesized PCR primers and sequenced DNA. BLAST searches were done at the National Center for Biotechnology Information Web site using standard proteinprotein BLAST (blastp). Alignments were done using multiple sequence alignment with hierarchical clustering (Corpet, 1988) using the Multalin interface at the Institut National de la Recherche Agronomique Web site and the resulting alignments were formatted using Multiple Align Show from the Sequence Manipulation Suite available at the University of Alberta Web site. Transketolase signature sequences I and II were identified using ScanProsite on the ExPASy Molecular Biology Server. The GC contents of the Rv2682c and Rv3379c were determined at the M. tuberculosis Structural Genomics Consortium Web site.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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