Biochemical properties of MutT2 proteins from Mycobacterium tuberculosis and M. smegmatis and their contrasting antimutator roles in Escherichia coli.
Mycobacterium 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-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (Km and Vmax) 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.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Pau Biak
PB
Sang
Umesh
U
Varshney
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D011994:Recombinant Proteins; C078206:8-oxodeoxyguanosine triphosphate; C024107:2'-deoxycytidine 5'-triphosphate; C057674:8-hydroxyguanosine triphosphate; C034817:5-methyldeoxycytidine triphosphate; D006160:Guanosine Triphosphate