Biochemical properties of MutT2 proteins from Mycobacterium tuberculosis and M. smegmatis and their contrasting antimutator roles in Escherichia coli

Abstract

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.

Fig 1

(A) Sequence comparison of EcoMutT, MtuMutT2, and MsmMutT2. The amino acid sequences were aligned by using the ClustalW program and the BOX SHADE (version 3.21) server. Identical residues are shown in black, whereas similar residues are shown in light gray boxes. The Nudix box sequence motif is underlined. (B) Analysis of purified MutT proteins (∼3 μg each) on 17% SDS-polyacrylamide gels. Lanes: 1, protein size markers (M; as indicated); 2, MtuMutT2; and 3, MsmMutT2.

Fig 2

HPLC separation of the substrates (dNTPs) and products (deoxynucleoside monophosphates) formed by the action of MtuMutT2. All dNTPs were taken at a concentration of ∼250 μM with 100 ng of the enzyme, and the reaction was done at 37°C for 10 min. The product and substrates were separated by HPLC using a C₁₈ column (Acclaim C₁₈, 120A, 3 μm, 4.6 by 150 mm) in an isocratic flow of 73 mM KH₂PO₄, 5 mM tetrabutylammonium hydroxide, and 25% methanol at a flow rate of 0.5 ml min⁻¹. The retention times of the peaks (in minutes) are shown on the x axes, and the peak intensities in milli-absorbance units (mAU) are shown on the y axes. (A) Activity of MtuMuT2 on different dNTP substrates at 0 min and 10 min: 8-oxo-dGTP (i), 8-oxo-GTP (ii), dCTP (iii), and 5-Me-dCTP (iv). (B) Activity of MtuMuT2 on mixtures of different dNTP substrates at 0 min and 10 min: mixture of 8-oxo-dGTP and dCTP (i) and mixture of 8-oxo-dGTP, dGTP, dATP, dCTP, and dTTP (ii). WVL, wavelength.

Fig 3

HPLC separation of the substrates (dNTPs) and products (deoxynucleoside monophosphates) formed by the action of MsmMutT2 and EcoMutT. The products and substrates were separated by HPLC using s DNAPac PA200 analytical column (4 by 250 mm) in buffer consisting of 25 mM Tris-HCl (pH 9.0) and a gradient of 1 M LiCl from 0% to 40% for 25 min at a flow rate of 0.5 ml min⁻¹. The retention times of the peaks (in minutes) are shown on the x axes, and the peak intensities in milli-absorbance units are shown on the y axes. The activities of MsmMutT2 on 8-oxo-dGTP (i), 8-oxo-dGTP with EcoMutT as a control (ii), dCTP (iii), and 5-Me-dCTP (iv) are shown.

Fig 4

Michaelis-Menten plot of the kinetics of MtuMutT2 (A) and MsmMutT2 (B) on 8-oxo-dGTP and dCTP. The velocity at each substrate concentration was calculated from an independent time course reaction using 5 to 8 time points.

Fig 5

Mutation frequency of E. coli MG1655 and its ΔmutT::kan derivative harboring plasmid pBADHisB or its derivatives harboring Eco-mutT, Msm-mutT2, and Mtu-mutT2. Mutation frequency was calculated by dividing the number of colonies that appear on LB agar containing rifampin by the number of colonies that appear on LB agar. The values are the means ± SDs of 6 independent colonies.

Fig 6

Frequency of Lac⁺ reversion of E. coli CC101, its ΔmutT::kan derivative harboring plasmid pBADHisB, or its derivatives harboring Eco-mutT, Msm-mutT2, and Mtu-mutT2 to score for A-to-C mutations. Reversion frequencies were calculated by dividing the number of colonies that appear on a minimal lactose plate by the number of colonies that appear on a minimal glucose plate. The data are represented as means ± SDs of 10 independent colonies.