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 substitutio n at the rarely attacked beta-P, to yield NMP and pyrophosphate. Previ ous heteronuclear NMR studies of MutT have shown the secondary structu re to consist of a five-stranded mixed beta-sheet connected by the loo p I-alpha-helix I-loop II motif, by two tight turns, and by loop III, and terminated by loop IV-alpha-helix II [Abeygunawardana et al. (1993 ) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 1 3081-13087). Complete side-chain assignments of H-1 and C-13 resonance s 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 N-15 -resolved NOESY-HSQC and 3D C-13-resolved NOESY-HSQC spectra, includin g 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints a nd 34 backbone hydrogen bond restraints were used to determine the ter tiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globula r and compact with the parallel portion of the beta-sheet sandwiched b etween the two alpha-helices, forming an alpha + beta fold. The essent ial divalent cation has previously been shown to bind near residues Gl y-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to b ind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick e t al. (1995) Biochemistry 34, 5577-5586]. The tertiary structure of Mu tT 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, 5 6, 57, and 98) form a patch of strongly negative electrostatic potenti al Likely constituting the metal binding site. This site is contiguous with a deep cleft between beta-strands A, C, and D and loop I which m ay contribute to the nucleotide binding site. This location of the act ive site is consistent with mutagenesis studies and with sequence homo logies among MutT-like pyrophosphohydrolases.