Biochemical Properties of MutT2 Proteins from Mycobacterium tuberculosis and M. smegmatis and Their Contrasting Antimutator Roles in Escherichia coli

Abstract

bMycobacterium tuberculosis, the causative agent of tuberculosis, is at increased risk of accumulating damaged guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP because of its residency in the oxidative environment of the host macrophages. By hydrolyzing the oxidized guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role in allowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we purified recombinant M. tuberculosis MutT2 (MtuMutT2) and M. smegmatis MutT2 (MsmMutT2) proteins from M. tuberculosis (a slow grower) and M. smegmatis (fast growing model mycobacteria), respectively, for their biochemical characterization. Distinct from the Escherichia coli MutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxodGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (K m and V max ) revealed that while MtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP, MsmMutT2 hydrolyzes them nearly equally. Also, MsmMutT2 is about 14 times more efficient than MtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP. Consistent with these observations, MsmMutT2 but not MtuMutT2 rescues E. coli for MutT deficiency by decreasing both the mutation frequency and A-to-C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins.

A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

“…Consistent with this, a double deletion mutant defective for both mutM and mutY displays a higher mutation rate (G·C → T·A transversions) than the respective single deletion mutants [21,34,35]. Further deletion of mutT in this background did not enhance this effect [30] and underscores the specific role for MutT in repairing 8-oxoGua in the nucleotide pool prior to replication [36].…”

Structural and kinetic studies on adenylosuccinate lyase from Mycobacterium smegmatis and Mycobacterium tuberculosis provide new insights on the catalytic residues of the enzyme

“…A comparison of the k cat for the cleavage of SAMP in all the kinetically characterized bacterial ASLs shows an inverse relationship with the corresponding doubling time ( Table 5). Similar differences in the efficiency of the enzymes from Mycobacteria and other organisms as a result of differences in growth rates have been reported previously [33,34]. Furthermore, the mycobacterial genomes have a high GC content of~65% and hence their requirement of adenine nucleotides required for DNA or RNA synthesis is correspondingly low.…”

Division of labor amongMycobacterium smegmatisRNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase

“…Are there not other means of preventing or dealing with oxidized ribo lesions? Mycobacteria have MutT-type enzyme systems that can detoxify oxo-dGTP and oxo-rGTP in the NTP pool, by converting them to oxo-dGMP and oxo-rNMP (34,35). Whether the M. smegmatis MutT systems are operative in stationary phase at a level sufficient to cope with peroxide stress on the guanine nucleotide pool is not clear.…”