Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) Proteins Constitute a Two-stage Mechanism of 8-Oxo-dGTP and 8-Oxo-GTP Detoxification and Adenosine to Cytidine Mutation Avoidance*

Background: MutT proteins hydrolyze oxidatively damaged nucleotides. Results: Mycobacterium tuberculosis MutT1 and Rv1700 cooperate to detoxify 8-oxo-dGTP to 8-oxo-dGMP and 8-oxo-GTP to 8-oxo-GMP and protect bacteria against oxidative stress. Conclusion: MtuMutT1 and Rv1700 constitute a novel two-stage mechanism of 8-oxo-dGTP and 8-oxo-GTP detoxification. Significance: Functional homolog of mycobacterial MutT, whose co-targeting may accentuate the impact of the known antibiotics, has been identified and characterized. Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a Km of ∼50 μm and Vmax of ∼0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a Km of ∼9.5 μm and Vmax of ∼0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.

Mycobacterium tuberculosis causes tuberculosis, which is one of the most feared infectious diseases of humankind (1). The pathogen resides and multiplies within the host macrophages where it is subjected to the reactive oxygen species and reactive nitrogen intermediates released as part of the host's innate immune response (2). Because guanine and cytosine are highly susceptible to damage by reactive oxygen species and reactive nitrogen intermediates, the GϩC richness of the M. tuberculosis genome makes it highly prone to these agents.
A common damage that occurs due to oxidative stress is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxo-G), 4 both in DNA and the free nucleotide pool. Although 8-oxo-G in DNA does not block replication of DNA, it induces base mismatches resulting in C to A mutations, and incorporation of 8-oxo-G against A in the template causes A to C mutations. On the other hand, incorporation of 8-oxo-G in RNA leads to mistranslation of proteins, which may lead to cell death (3)(4)(5)(6)(7)(8). Like other organisms, mycobacteria possess an elaborate GO repair system consisting of Fpg (MutM), MutY, and MutT enzymes to deal with the oxidative threats to genomic integrity (9 -11). MutT is a Nudix hydrolase protein characterized by a 23amino acid conserved sequence motif named Nudix box, GX 5 EX 7 REUXEEXGU, where U is a bulky hydrophobic residue and X is any residue (12). In Escherichia coli, MutT hydrolyzes 8-oxo-dGTP and 8-oxo-dGDP to 8-oxo-dGMP. It also hydrolyzes 8-oxo-GTP and 8-oxo-GDP to 8-oxo-GMP. Thus, the MutT activities prevent genomic mutations and maintain the fidelity of protein synthesis under oxidative stress (13,14).
Bioinformatics analyses predicted the presence of nine proteins with Nudix hydrolase motif in mycobacteria (18,19). Of these, four have been annotated as MutT homologs (MutT1, MutT2, MutT3, and MutT4). Here, we report the biochemical characterization and physiological role of MutT1 of M. tuberculosis (MtuMutT1) and another Nudix box protein, Rv1700. Our results suggest that in mycobacteria, the MtuMutT1 and Rv1700 function in concert to detoxify 8-oxo-dGTP to 8-oxo-dGMP and play an important role in supporting cellular growth under oxidative stress.

EXPERIMENTAL PROCEDURES
Plasmids, Bacterial Strains, and Media-Plasmids and bacterial strains used are listed in Table 1. dNTPs were purchased from Jena Bioscience. Unless specified otherwise, E. coli was grown in Luria-Bertani (LB) broth (Difco). Agar (1.5%) was added to the LB broth for growth on solid surface (LB-agar). Culture media were supplemented with ampicillin (Amp, 100 g/ml), tetracycline (7.5 g/ml), respectively, as required.
Cloning of MtuMutT1-The open reading frame (ORF) sequence of MtuMutT1 (Rv2985) was retrieved from the M. tuberculosis database and PCR-amplified from M. tuberculosis H37Rv DNA using MtuMutT1 Fp (5Ј-ggagttcatatgtcgatccagaactcggtc-3Ј) and MtuMutT1 Rp (5Ј-atatgatcgattttaggcccg-cacgttggcgg-3Ј) primers containing NdeI and ClaI sites, respectively. PCR was carried out using Dynazyme EXT DNA polymerase (Finnzymes). The reaction was heated to 94°C for 4 min followed by 30 cycles of incubations at 94°C for 1 min, 58°C for 30 s, and 72°C for 1 min, and then incubated at 72°C for 10 min. The amplicon (957 bp) was digested with NdeI and ClaI and cloned into similarly digested pET14b (isolated from E. coli JM110 (dam Ϫ and dcm Ϫ strain)) to obtain pET14bMtuMutT1. The clone was confirmed by HincII digestion and DNA sequence analysis. MtuMutT1 was then subcloned from pET14bMtuMutT1 into pBAD-HisB by partially digesting with NcoI and EcoRI (due to the presence of an internal EcoRI site), taking along the His 6 tag from pET14b vector.
Cloning of Rv1700-Rv1700 (ADPRase) ORF was amplified by PCR from M. tuberculosis H37Rv genomic DNA using Rv1700 Fp (5Ј-cgtcatatggctgagcatgatt-3Ј) and Rv1700 Rp (5Јtgcaagcttcttcatcgctcg 3Ј) primers containing NdeI and HindIII sites, respectively. PCR was carried out using the Phusion DNA polymerase by first heating the tube at 98°C for 4 min followed by 36 cycles of incubations at 98°C for 1 min, 63°C for 30 s, and 72°C for 45 s, and a final incubation of 10 min at 72°C. The amplicon (637 bp) was eluted, ligated to pJET1.2 vector, and transformed into E. coli TG1 to obtain pJET Rv1700. The pJET Rv1700 was then digested with NdeI and HindIII, and the insert was ligated into similarly digested pTrcNdeHis to generate pTrcRv1700. The pTrcRv1700 was confirmed by restriction digestion with EcoRV and DNA sequencing.
Purification of MtuMutT1-A single colony of E. coli MG1655 ⌬mutT/pBADMtuMutT1 was inoculated in 20 ml of LB broth containing Amp and grown for ϳ4 h at 37°C (A 600 ϳ0.4 -0.6). This culture was diluted 1,000-fold to grow 6 liters of culture in LB broth containing Amp at 18°C. When the culture reached an A 600 of ϳ0.6, expression of MtuMutT1 was induced with 0.02% arabinose, and the culture was further incubated for 3 h at 18°C. The cells were harvested, washed, and resuspended in 50 ml of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% glycerol (v/v), 2 mM ␤-mercaptoethanol) containing 20 mM imidazole, ultrasonicated, and centrifuged at 12,000 rpm for 1 h using AF-5004CA rotor in Kubota 3500. The supernatant was then centrifuged at 29,000 rpm for 2 h in (Avanti TM J-30I, JA 30.50 Ti). The supernatant was loaded onto Purification of Rv1700-The plasmid pTrcRv1700 was transformed into E. coli JW0097⌬mutT. A single colony was inoculated into 50 ml of LB broth (Amp), grown at 37°C to A 600 of ϳ0.6, and subcultured into 4 liters of LB broth containing Amp, supplemented with 0.3 mM isopropyl 1-thio-␤-D-galactopyranoside when the culture reached an A 600 of ϳ0.6, and allowed to grow for 2.5 h. The culture was harvested and the cells resuspended into buffer B (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol (v/v) and 2 mM ␤-mercaptoethanol) containing 10 mM imidazole. Cells were lysed by ultrasonication, processed for S100 extract preparation and purification from Ni-NTA column as above for MtuMutT1. The fractions containing Rv1700 protein were pooled, concentrated using Centricon tubes (30-kDa cutoff), and dialyzed against 50% glycerol in buffer B at 4°C.
Activity Assays for MtuMutT1, Rv1700, and EcoMutT-Various NTPs and dNTPs (ϳ250 M) were incubated with 100 ng of MtuMutT1, Rv1700, or EcoMutT in a 10-l reaction containing 1ϫ MutT buffer (20 mM Tris-HCl, pH 8.0, 8 mM MgCl 2, 40 mM NaCl, 5 mM DTT, 2% glycerol) for 30 min at 37°C (for kinetic analysis 8-oxo-dGTP was used as substrate at 12.5-125 M concentrations, and the reaction time was 10 min). The reactions were stopped with 0.1% SDS and analyzed by HPLC (UltiMate 3000) using a DNAPac column (DNAPac PA200 analytical, 4 ϫ 250 mm) using solvent system consisting of 25 mM Tris-HCl, pH 9.0, and 1 M lithium chloride gradient (0 -40%) at a flow rate of 0.5 ml/min, for 25 min. For kinetic analysis of Rv1700, 8-oxo-dGDP was used at a 5-40 M concentration, and the reactions were terminated after a 15-min incubation. The K m and V max were determined from Michaelis-Menten plots.
Mass Spectrometry Analysis-For MALDI-MS analysis of the reaction products, a 5-l reaction consisting of 10 ng of purified protein (MtuMutT1, EcoMutT, or Rv1700) and 1.5 nmol of substrate (8-oxo-dGTP, 8-oxo-dGDP, or 8-oxo-GTP) was incubated at 37°C overnight (except for the combined reaction of MtuMutT1 and Rv1700 which was done for 2 h) in a buffer containing 20 mM Tris-HCl, pH 7.5, 8 mM MgCl 2 , 5 mM DTT, 40 mM NaCl, and 2% glycerol, mixed with equal volume of ␣-cyanocinnamic acid (10 mg/ml in 50% acetonitrile ϩ 0.1% trifluoroacetic acid) and spotted directly on MALDI plates. Spectra were acquired in positive ion mode using a Bruker-Daltonics AutoFlex III SmartBeam mass spectrometer using a 337-nm laser source, and data were analyzed in FlexAnalysis software provided by the manufacturer. Reaction products were identified by comparison with masses of standards.
Survival Analysis-Various plasmid constructs were transformed into E. coli MG1655 and its ⌬mutT derivative. Three independent colonies were inoculated in LB broth and grown to A 600 of ϳ0.6. Cells were harvested and washed with 0.1 mM phosphate-buffered saline, PBS (pH 7.0). The cells were resuspended in PBS at A 600 ϳ0.2. Different concentrations of H 2 O 2 (0, 1, and 2 mM) were added to the cell suspensions and plated on LB-agar at 0, 1, 2, and 3 h of incubation at 37°C to determine total viable counts as cfu. The log values of cfu were plotted. For treatment with acidified NaNO 2 , cells were washed with PBS adjusted to pH 5.5, and experiments were done as described above. NaNO 2 concentrations of 0 and 7.5 mM were used.
Analysis of Mutation Spectrum-Various plasmid constructs were introduced into E. coli CC101-106 and their ⌬mutT derivatives by transformation. In the case of E. coli CC101-106 strains, isolated colonies were streaked on LB-agar plates containing X-gal (50 g/ml), and single white colonies were inoculated into 2 ml of LB broth and grown at 37°C for 14 h. Aliquots (100 l) of the 10 Ϫ6 dilution of the cultures were plated on M9 minimal media agar containing 0.2% glucose for viable count determination, and 1-ml cultures were spun down and plated on minimal media agar containing 0.2% lactose to determine Lac ϩ revertants. Plates were incubated at 37°C. The number of revertants and viable counts was enumerated after 24 and 72 h, respectively.

Purification and Activity Assays of MtuMutT1-MtuMutT1
ORF was cloned in a T7 RNA polymerase based expression vector, pET14b, and checked for MtuMutT1 expression at different temperatures (37, 30, and 18°C) as well as with different concentrations (0.3-0.6 mM) of isopropyl 1-thio-␤-D-galactopyranoside. In these conditions, MtuMutT1 partitioned into insoluble fraction of the cell lysate. Subsequently, MtuMutT1 ORF was subcloned into pBADHisB, and expression of Mtu- MutT1 was checked at 37, 30, and 18°C upon induction with arabinose (0.02%). Expression from the pBADMtuMutT1 at 18°C yielded MtuMutT1 in S100 supernatant, which was subjected to purification by Ni-NTA and gel filtration chromatography. The gel filtration chromatography profile (Fig. 1A) showed that on the SDS-PAGE, MtuMutT1 migrated as a doublet. A second chromatography of the pooled fractions from gel filtration column on Ni-NTA column still retained the doublet character of MtuMutT1 on SDS-PAGE (Fig. 1B). MALDI-MS analysis showed that both bands represent MtuMutT1, suggesting that the MtuMutT1 was pure. Further, an analysis of the eluted proteins showed redistribution of the protein from each band into the upper and lower bands, suggesting that the doublet character of MtuMutT1 could be due to the presence of a faster migrating minor conformer (supplemental Fig. S1). Biochemical activity of MtuMutT1 was analyzed using dGTP, GTP, dATP, ATP, dCTP, CTP, 8-oxo-dATP, 8-oxo-dGTP, and 8-oxo-GTP. Among these, MtuMutT1 used 8-oxo-dGTP and 8-oxo-GTP as substrates. Unlike EcoMutT, which hydrolyzed 8-oxo-dGTP to 8-oxo-dGMP ( Fig. 2A, compare panel v with the standards in panels i-iii), MtuMutT1 hydrolyzed it to 8-oxo-dGDP (compare panel iv with panels i-iii and v). Likewise, the retention time of the product of MtuMutT1 reaction on 8-oxo-GTP suggests that 8-oxo-GTP was also hydrolyzed to the corresponding nucleoside diphosphate (Fig. 2B, compare panel ii with panels i (an 8-oxo-GTP standard) and iii (8-oxo-GMP, a known reaction product of EcoMutT on 8-oxo-GTP)).
Purification and Activity Assays Using Rv1700-As the products of MtuMutT1-mediated reaction 8-oxo-dGDP and 8-oxo-GDP (from 8-oxo-dGTP and 8-oxo-GTP, respectively) can be converted back to 8-oxo-dGTP and 8-oxo-GTP (for example by nucleoside diphosphate kinase), they would still compromise the fidelity of DNA and RNA synthesis, respectively. Therefore, we explored if 8-oxo-dGDP and 8-oxo-GDP may be further converted to nucleoside monophosphates by another protein. Such Nudix box proteins (NUDT5, NUDT18 (MTH3)) have

FIGURE 2. Activity assays of MtuMutT1, EcoMutT, and Rv1700 proteins on 8-oxo-dGTP/8-oxo-dGDP (A) and 8-oxo-GTP (B).
Approximately 250 M 8-oxo-G nucleotides were incubated with~100 ng of MtuMutT1, Rv1700, and EcoMutT, as indicated in a 10-l reaction in 1ϫMutT buffer for 30 min at 37°C. The reactions were stopped with 0.1% SDS and analyzed by HPLC using a DNAPac column with buffer A consisting of 25 mM Tris-HCl, pH 9.0, and 1 M lithium chloride as buffer B. Lithium chloride gradient (0 -40%) was set at a flow rate of 0.5 ml/min for 25 min. Various panels are as indicated.
been described in human (20). Hence, we searched for a homolog of NUDT5/NUDT18 among the Nudix hydrolases in M. tuberculosis and identified Rv1700 as a candidate. The ORF of Rv1700 protein (annotated as ADPRase (21) was cloned into pTrcNdeHis expression vector, and the protein was purified to apparent homogeneity by Ni-NTA chromatography (Fig. 1B). Analysis of biochemical activity of this protein revealed that it indeed converted 8-oxo-dGDP to 8-oxo-dGMP ( Fig. 2A, panel  vi). In concert with MtuMutT1, it (Rv1700) converted the reaction intermediate of 8-oxo-dGDP to 8-oxo-dGMP ( Fig. 2A,   panel vii). However, it (Rv1700) did not hydrolyze 8-oxo-dGTP ( Fig. 2A, panel viii). Likewise, Rv1700 converted 8-oxo-GDP to 8-oxo-GMP (Fig. 2B, panel iv) but did not act on 8-oxo-GTP (Fig. 2B, panel v). We may add that a peak of retention time ϳ2.5 min and a general background in the initial stages of the elution seen in the enzyme reactions corresponded to UV-absorbing contaminants in the proteins plus buffer (Fig. 2B, panel  vi). Further, the 8-oxo-dGTP and 8-oxo-GTP preparations contained detectable but minor peaks corresponding to 8-oxo-dGDP and 8-oxo-GDP, respectively. Because the area under these peaks did not change in Fig. 2A (panel viii) or Fig. 2B  (panel v), the presence of these minor peaks in these panels was ignored.
Mass Spectrometric Analyses-To validate further the nature of the products of 8-oxo-dGTP/8-oxo-GTP treatment with MtuMutT1/Rv1700, we analyzed the reaction products by MALDI-MS. Despite the fact that background peaks are typically present in the lower mass range due to ionization of matrix and low molecular mass impurities, the m/z signals of 524.095, 444.015, and 364.057 corresponding to the characteristic theoretical masses of 523.99, 444.02, and 364.06, respectively, for the standards of 8-oxo-dGTP, 8-oxo-dGDP, and 8-oxo-dGMP were distinctively obtained (Fig. 3, panels i-iii, as indicated by the arrows), making it possible to analyze the reaction products by this method. On treatment with MtuMutT1, 8-oxo-dGTP was converted into 8-oxo-dGDP (m/z peak of 444.023), and no peaks corresponding to 8-oxo-dGMP were seen (Fig. 3, panel  iv). As a control, treatment of 8-oxo-dGTP with EcoMutT resulted in its conversion to 8-oxo-dGMP (m/z peak of 364.034) (Fig. 3, panel v). Likewise, the peak corresponding to 8-oxo-dGMP was also seen when 8-oxo-dGTP was treated simultaneously with MtuMutT1 and Rv1700 (m/z peak of 364.069, panel vi) or upon treatment of 8-oxo-dGDP with Rv1700 alone (m/z peak of 364.066, panel vii). Also, as expected, treatment of 8-oxo-dGTP (m/z peak of 524.011, panel viii) with Rv1700 resulted in production of neither 8-oxo-dGDP nor 8-oxo-  Fig. S2, panels i-vi), the results shown in Fig. 3 (panels i-vii) further validate the observations made in Fig. 2A. Similar anal-yses of the reaction products of 8-oxo-GTP (theoretical mass 539.99) with MtuMutT1 and Rv1700 were complicated by the presence of matrix peaks (m/z 380.08) corresponding to the theoretical mass of 8-oxo-GMP (380.05) (supplemental Fig.  S3). Nonetheless, consistent with the results shown in Fig. 2B, treatment of 8-oxo-GTP with MtuMutT1 resulted in the disappearance of an m/z peak of 540.126 seen in the 8-oxo-GTP standard and the appearance of a peak of m/z 459.992 corresponding to 8-oxo-GDP with a predicted mass of 460.02 (Fig. 3,  panels ix and x). However, for the reason mentioned above, and for the lack of standards of 8-oxo-GDP and 8-oxo-GMP, the MS analyses of the reaction products of 8-oxo-GTP were not pursued any further.
Analysis of Kinetic Parameters of Substrate Utilization by MtuMutT1 and Rv1700-The kinetics of 8-oxo-dGTP to 8-oxo-dGDP conversion by MtuMutT1 and 8-oxo-dGDP to 8-oxo-dGMP by Rv1700 were determined using HPLC (Fig. 4). Effect of H 2 O 2 and Acidified NaNO 2 on Survival-One of the major sources of oxidative damage to nucleotide pool is reactive oxygen species. Hence, we treated various strains of E. coli with H 2 O 2 to analyze the effect of the oxidative stress and the impact of MtuMutT1/Rv1700 on their survival. As a control (Fig. 5A, panel i), we observed no apparent differences in the survival of the untreated E. coli MG1655, and its ⌬mutT derivatives harboring vectors alone (pACDH/pTrcNdeHis) or the expression constructs for the two proteins (pACDHMtuMutT1/ pTrcRv1700). Because isopropyl 1-thio-␤-D-galactopyranoside was not used, MtuMutT1 and Rv1700 were expressed at low levels (undetectable by Coomassie Blue staining following SDS-  PAGE, supplemental Fig. S4). The survival of the ⌬mutT strain harboring vectors alone (curve 2) was compromised upon treatment with 1 mM H 2 O 2 and severely compromised upon treatment with 2 mM H 2 O 2 for 3 h (panels ii and iii, respectively). When treated with 1 mM H 2 O 2 , the presence of Mtu-MutT1 and/or Rv1700 rescued strain survival (panel ii). In the experiment with 2 mM H 2 O 2 , the simultaneous presence of MtuMutT1 and Rv1700 best rescued the strain survival. In fact, their simultaneous presence conferred tolerance to H 2 O 2 treatment in that the strain harboring the expression constructs for MtuMutT1 and Rv1700 showed better survival than even the parent strain (panel iii, compare curve 5 with curve 1). As shown in Fig. 5B, we did not observe any specific detrimental effect upon treatment of the ⌬mutT strain with acidified NaNO 2 even at a concentration as high as 7.5 mM. Survival of all strains was equally compromised (panels i and ii), suggesting that MutT deficiency did not cause any specific compromise of survival under the acidified NaNO 2 , nor did the presence of either or both of the proteins (MtuMutT1 and Rv1700) offer any advantage to the strains under the conditions used.
Rescue of A to C Mutations in E. coli by MtuMutT1 and Rv1700-E. coli strains have been characterized which possess specific mutations at the active site Glu 461 codon (GAG) of the lacZ gene to TAG, GGG, CAG, GCG, GTG, or AAG in strains CC101, CC102, CC103, CC104, CC105, and CC106, respectively, which fail to grow on minimal lactose plates (22). Conditions that result in increase in specific mutations, e.g. A to C (or T to G) in CC101, result in the appearance of isolated colonies on minimal lactose because of increased chances of TAG (CTA) to GAG (CTC) reversion of the lacZ mutation. Introduction of a knock-out allele of mutT (known to result in increase in A to C transversion mutations) in these strains results in a tremendous increase in the number of colonies of E. coli CC101 (but not in the case of other strains) coming up on minimal lactose medium. Plating of E. coli CC101⌬mutT on minimal lactose agar, therefore, allows one to investigate whether MtuMutT1 and Rv1700 decrease the frequency of appearance of these colonies on minimal lactose plates. As shown in Fig. 6, E. coli CC101⌬mutT resulted in 2905/10 8 colonies on minimal lactose plates (bar 2). Although there was a decrease in the frequency of colonies appearing in the presence of either MtuMutT1 or Rv1700 (bars 3 and 4), the best rescue was observed by their simultaneous presence (bar 5). As a control, when we checked for the impact of deletion of mutT (⌬mutT) in CC102-106 strains, there were no detectable increases in reversion of the mutant lacZ (data not shown).

DISCUSSION
Recent studies in E. coli have highlighted the importance of MutT proteins in protection against oxidative stress leading to generation of oxidatively damaged forms of guanine in dGTP and GTP. In fact, treatment of bacteria even with antibiotics whose primary targets are known to be other essential cellular processes may cause toxicity via oxidative damage to GTP/ dGTP and their misincorporation in DNA and RNA. Interestingly, MutT overexpression was found to rescue the DNAdamaging effects of the antibiotics such as ␤-lactams and quinolones (23). Thus, inhibiting MutT proteins in bacteria may provide with a strategy to accentuate the impact of the known antibiotics.
M. tuberculosis, the causative agent of tuberculosis, is a highly successful pathogen, which resides latently in nearly one third of the human population (1). Appearance of multidrugresistant, extremely drug-resistant, and more recently of totally drug-resistant strains of M. tuberculosis has made the management of tuberculosis a serious health concern (24). The primary site of infection of M. tuberculosis is the alveolar macrophages (25). As a part of host's innate immune response, the macrophages respond by producing reactive oxygen species and reactive nitrogen intermediates (2). The oxidative damage causes accumulation of damaged guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. As the 8-oxo-dGTP is a potent mutagen (because of its ambiguous base pairing properties), the MutT proteins are likely to be crucial for M. tuberculosis survival in the host. As shown in Fig. 5, MutT deficiency in E. coli results in high susceptibility to oxidative stress; also, in mice, MutT deficiency has been reported to result in increased occurrence of tumors of various organs (26).
A functional homolog of E. coli MutT had not been characterized so far from M. tuberculosis. MtuMutT2 converts 8-oxo-dGTP to 8-oxo-GMP (27). However, it is known to be a better dCTPase (18). Moreover, it is kinetically very inefficient both as 8-oxo-dGTPase and dCTPase, and it does not rescue E. coli for its deficiency of MutT activity (27). Therefore, it appears that MtuMutT2 may not be a major player in vivo, at least not as an 8-oxo-dGTPase. Genetic studies have also shown that the MutT2 deficiency in M. tuberculosis as well as in Mycobacterium smegmatis causes an insignificant increase of ϳ1.5-fold in mutation frequency, as assessed by appearance of Rif R colonies (28).
In this study, we have shown that MtuMutT1 functions as an efficient 8-oxo-dGTPase. However, it has a novel activity of converting 8-oxo-dGTP to 8-oxo-dGDP. MutT proteins are commonly known to convert either a nucleoside triphosphate or nucleoside diphosphate to nucleoside monophosphate (see the Introduction). To convert 8-oxo-dGDP, the product of MtuMutT1, to 8-oxo-dGMP, the activity of another Nudix box hydrolase, Rv1700, is required (Figs. 2 and 3). Together, these FIGURE 6. Mutation frequency analysis with respect to A to C (or T to G) using E. coli CC101 strain and its ⌬mutT derivative harboring vector alone or the expression constructs of MtuMutT1 and/or Rv1700 by scoring for Lac ؉ revertants on minimal media containing lactose as carbon source. Mutation frequency was calculated by dividing the number of Lac ϩ revertants with the number of bacteria (colonies) on minimal glucose plate. The mean Ϯ SD of (n ϭ 9) independent colonies were calculated (shown above the bars) and plotted relative to E. coli CC101.
two proteins provide maximum protection to the MutT-deficient E. coli against oxidative damage both for its survival as well as prevention of A to C mutations (Figs. 5 and 6). Rv1700 has earlier been shown to possess ADPRase activity (21). Our studies show that this protein has additional activity of converting 8-oxo-dGDP and 8-oxo-GDP to 8-oxo-dGMP and 8-oxo-GMP, respectively. It should also be said that our observations do not rule out the possibility of some of the uncharacterized MutT (MtuMutT3, MtuMutT4) or other Nudix box proteins carrying out the function of efficiently hydrolyzing 8-oxo-dGTP and 8-oxo-GTP directly to their corresponding monophosphates. However, the fact that of the four MutT proteins described in mycobacteria, deficiency of MutT1 has the maximum mutator phenotype of approximately 15-fold in mycobacteria (28) suggests that MutT1 carries out the major MutT function. Thus, it appears that even in M. tuberculosis cells, MutT1 initiates the major function of detoxifying oxidatively damaged guanine nucleoside triphosphates. Both MtuMutT1 and Rv1700 proteins are highly conserved among mycobacteria (supplemental Figs. S5 and S6). However, it is unclear what advantage such a two-stage mechanism of detoxifying oxidatively damaged 8-oxo-dGTP/8-oxo-GTP may offer. Based on the sequence comparisons, we identified that the mycobacterial MutT1 proteins have two domains. Of these, the N-terminal Nudix hydrolase domain corresponds to the single-domain E. coli MutT (29) whereas the C-terminal domain corresponds to a phosphatase domain belonging to the histidine phosphatase superfamily proteins (30). We are currently in the process of understanding the three-dimensional structure of M. smegmatis MutT1 using x-ray crystallography. Insights from these studies may provide us with a better understanding of the substrate specificity of mycobacterial MutT1.