An unusual activity of mycobacterial MutT1 Nudix hydrolase domain as a protein phosphatase regulates nucleoside diphosphate kinase function

MutT proteins are Nudix hydrolases characterized by the presence of a Nudix box, GX5EX7REUXEEXGU, where U is a bulky hydrophobic residue and X is any residue. Major MutT proteins hydrolyze 8-oxo-(d)GTP (8-oxo-GTP or 8-oxo-dGTP) to the corresponding 8-oxo-(d)GMP, preventing their incorporation into nucleic acids. Mycobacterial MutT1 comprises an N-terminal domain (NTD) harboring the Nudix box motif, and a C-terminal domain (CTD) harboring the RHG histidine phosphatase motif. Interestingly, unlike other MutTs, the MutT1 hydrolyses the mutagenic 8-oxo-(d)GTP to the corresponding 8-oxo-(d)GDP. Nucleoside diphosphate kinase (NDK), a conserved protein, carries out reversible conversion of (d)NDPs to (d)NTPs through phospho-NDK (NDK-<i>Pi</i>) intermediate. Recently, we showed that NDK-<i>Pi</i> converts 8-oxo-dGDP to 8-oxo-dGTP and escalates A to C mutations in a MutT-deficient <i>Escherichia coli</i>. We now show that both <i>Mycobacterium tuberculosis</i> MutT1 and <i>Mycobacterium smegmatis</i> MutT1, through their NTD (Nudix hydrolase motifs) function as protein phosphatase to regulate the levels of NDK-<i>Pi</i> and prevent it from catalyzing conversion of (d)NDPs to (d)NTPs (including conversion of 8-oxo-dGDP to 8-oxo-dGTP). To corroborate this function, we show that <i>Msm</i>MutT1 decreases A to C mutations in <i>E. coli</i> under the conditions of <i>Eco</i>NDK overexpression.IMPORTANCEMutT proteins, having a Nudix box domain, hydrolyze the mutagenic 8-oxo-dGTP to 8-oxo-dGMP. However, mycobacterial MutT (MutT1) comprises an N-terminal domain (NTD) harboring a Nudix box, and a C-terminal domain (CTD) harboring an RHG histidine phosphatase. Unlike other MutTs, mycobacterial MutT1 hydrolyses 8-oxo-dGTP to 8-oxo-dGDP. Nucleoside diphosphate kinase (NDK), a conserved protein, converts 8-oxo-dGDP to 8-oxo-dGTP through phospho-NDK (NDK-<i>Pi</i>) intermediate and escalates A to C mutations. Here, we show that the mycobacterial MutT1 is unprecedented in that its NTD (Nudix box), functions as protein phosphatase to regulate NDK-<i>Pi</i> levels and prevents it from converting dNDPs to dNTPs (including 8-oxo-dGDP to 8-oxo-dGTP conversion). In addition, mycobacterial MutT1 decreases A to C mutations in <i>Escherichia coli</i> under the conditions of NDK overexpression.