Cellular metabolism contributes to the production of reactive nitrogen intermediates (RNI), and reactive oxygen species (ROS). Both RNI and ROS play a major role in oxidative damage of nucleotides within the genome and in their free pools [1], [2], [3], [4], [5]. Misincorporation of damaged nucleotides may compromise the fidelity of replication [6], and transcription [7]. Of all the DNA/RNA bases, guanine, because of its low redox potential, is the most susceptible base to oxidative damages. Oxidative damage of (d)GTP (indicating either GTP or dGTP) leads to the production of 8-oxo-7,8 dihydroguanosine triphosphate (8-oxo-(d)GTP) [8], [9], [10]. As 8-oxo-G can pair with C or A, it is highly mutagenic in the DNA. Misincorporation of 8-oxo-G can lead to AT to CG transversion mutations [11], [12], [13]. To prevent AT to CG mutations, the organisms possess an elaborate GO repair pathway comprising MutM, MutY, and MutT. MutM, an 8-oxo-G DNA glycosylase, removes 8-oxo-G paired against C in DNA, and MutY, an adenine DNA glycosylase, removes A paired against 8-oxo-dG in DNA [14], [15], [16], [17], [18]. MutT proteins belong to the Nudix hydrolase superfamily, which includes a diverse group of enzymes requiring Mg2+. MutT proteins possess a conserved Nudix box, GX5EX7REUXEEXGU where U is Leu, Ile or Val and X is any residue [19], [20], [21].

E. coli MutT (EcoMutT) is the most extensively studied MutT [19], [20], [22]. It is an efficient dGTPase, which acts on 8-oxo-(d)GTP and 8-oxo-(d)GDP and converts them to 8-oxo-(d)GMP [23]. This property of MutT prevents wrongful utilization of 8-oxo-dGTP and 8-oxo-GTP for DNA or RNA synthesis, respectively. Also, by acting on 8-oxo-(d)GDP, MutT prevents their conversion to respective nucleoside triphosphates by nucleoside diphosphate kinase (NDK). Earlier, we showed that NDK escalates A to C mutations in a MutT deficient E. coli [24]. E. coli MutT was reported to hydrolyse 8-oxo-dGTP to 8-oxo-dGMP three times faster than hydrolysing dGTP to dGMP [23], [25], [26], [27]. NMR spectroscopy, kinetics, and mutational studies helped in a better understanding of its mechanism of action [22], [28], [29], [30].

In mycobacteria, four homologues of MutTs were identified based on the presence of Nudix box. These proteins are Rv2985 (MutT1); Rv1160 (MutT2); Rv0413 (MutT3); and Rv3908 (MutT4) in M. tuberculosis (Mtb); and MSMEG_2390 (MutT1); MSMEG_5148 (MutT2); MSMEG_0790 (MutT3); and MSMEG_6927 (MutT4), in M. smegmatis (Msm) [31], [32], [33]. Of these, MtbMutT1 and MsmMutT1 hydrolyse 8-oxo-(d)GTP to 8-oxo-(d)GDP [21], [34]. Although it may be added that under non-physiological conditions of high salt, high enzyme concentrations, and prolonged incubations mycobacterial MutT1 does produce detectable levels of 8-oxo-(d)GMP [34]. However, the mycobacterial MutT1 decreases A to C mutations in E. coli CC101 ΔmutT, and plays an important role in bacterial survival under oxidative stress [21]. Mycobacterial MutT2 acts as an efficient dCTPase, and it hydrolyses dCTP and 5-methyl-dCTP to dCMP and 5-methyl-dCMP, respectively [35]. MutT3 (RenU) is an important enzyme for the redox homeostatic system and contributes to the survival of mycobacteria inside macrophages and in biofilm formation [36]. MutT4 was reported to have a dATPase activity and play an antimutator role [33], [37].

While both EcoMutT and mycobacterial MutT1 use 8-oxo-(d)GTP as a common substrate, the products of their enzymatic activities are different. So far, there are no reports on why mycobacterial MutT1 hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GDP (unlike to 8-oxo-(d)GMP by EcoMutT). It was shown that a sequence called phosphohydrolase module in E. coli (37GGX5EX7REUXEEXG59), that nearly fully overlaps with the Nudix box is the most crucial element for EcoMutT activity [34], [38]. This sequence (phosphohydrolase module) is represented by (65KGX5EX7REUXEEXG87) in M. smegmatis.

In this study, we carried out mutational analyses on EcoMutT and MsmMutT1, activity assays, and in vivo studies to address this question. Our studies show that Gly37 in EcoMutT and Lys65 in MsmMutT1 are the main determinants for the cleavage specificities of the respective enzymes.