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

Abstract

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 hydrolyse 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) harbouring the Nudix box motif, and a C-terminal domain (CTD) harbouring the RHG histidine phosphatase motif. Interestingly, unlike other MutTs, the MutT1 hydrolyses the mutagenic 8-oxo-(d)GTP to 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-Pi) intermediate. Recently, we showed that NDK-Pi converts 8-oxo-dGDP to 8-oxo-dGTP and escalates A to C mutations in a MutT deficient Escherichia coli. We now show that both Mycobacterium tuberculosis MutT1, and M. smegmatis MutT1, through their NTD (Nudix hydrolase motifs) function as protein phosphatase to regulate the levels of NDK-Pi to NDK and prevent it from catalysing conversion of (d)NDPs to (d)NTPs (including conversion of 8-oxo-dGDP to 8-oxo-dGTP). To corroborate this function, we show that MsmMutT1 decreases A to C mutations in E. coli under the conditions of EcoNDK overexpression. ImportanceMutT proteins, having a Nudix box domain, hydrolyse 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, mycobaterial 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-Pi) 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-Pi 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 Escherichia coli under the conditions of NDK overexpression.