Solution Structure of the MutT Enzyme, a Nucleoside Triphosphate Pyrophosphohydrolase

The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked β-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed β-sheet connected by the loop I-α-helix I-loop II motif, by two tight turns, and by loop III, and terminated by loop IV-α-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071 - 13080; Weber etal. (1993) Biochemistry 32, 13081 - 13087). Complete side-chain assignments of ¹H and ¹³C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D ¹⁵N-resolved NOESY-HSQC and 3D ¹³C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (ø) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the β-sheet sandwiched between the two α-helices, forming an α + β fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-5586]. The tertiary structure of MutT shows these residues to be near each other along the loop I-helix I region of the enzyme. A cluster of five glutamate residues (41, 53, 56, 57, and 98) form a patch of strongly negative electrostatic potential likely constituting the metal binding site. This site is contiguous with a deep cleft between β-strands A, C, and D and loop I which may contribute to the nucleotide binding site. This location of the active site is consistent with mutagenesis studies and with sequence homologies among MutT-like pyrophosphohydrolases.