Abstract
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.
Highlights
Purification and Activity Assays of MtuMutT1—MtuMutT1 open reading frame (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
MtuMutT1 ORF was subcloned into pBADHisB, and expression of Mtu
There was a 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
Summary
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. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 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. 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. In Escherichia coli, MutT hydrolyzes 8-oxo-dGTP and 8-oxo-dGDP to 8-oxo-dGMP. It hydrolyzes 8-oxo-GTP and 8-oxo-GDP to 8-oxo-GMP. The MutT activities prevent genomic mutations and maintain the fidelity of protein synthesis under oxidative stress [13, 14]
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