Solution Structure of the MutT Enzyme, a Nucleoside Triphosphate Pyrophosphohydrolase

Abstract

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 P-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed P-sheet connected by the loop I-a-helix I-loop I1 motif, by two tight tums, and by loop 111, and terminated by loop IV-a-helix I1 (4) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure ofMutT 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 P-sheet sandwiched between the two a-helices, forming an a f , ! ? 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, 5577-55861. 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 P-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.The MutT enzyme, a pyrophosphohydrolase of 129 residues, catalyzes the unusual hydrolysis of nucleoside and deoxynucleoside triphosphates (NTP) by nucleophilic substitution at the rarely attacked ,!?-phosphorus, yielding pyrophosphate and a nucleotide (NMP) as products (Bhatnagar et al., 1991;Weber et al., 1992). Like other enzymes which catalyze substitution at the electron-rich ,!?-phosphorus, this enzyme requires two divalent cations for activity (Frick et al., 1994). The biological role of this enzyme is to prevent ' This work was supported in part by National Institutes of Health Grants DK28616 (to A.S.M.), GM18649 (to M.J.B.), and GM36358 (which supports A.G.G.).A complete listing of the distance restraints derived from the NOE data has been deposited at the Brookhaven Protein Data Bank (Chemistry Department, Brookhaven National Laboratory, Upton, NJ) together with the atomic coordinates of the family of 15 acceptable structures (file name IMUT).

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

“…It therefore seems that recombinant YSA1H migrates anomalously on SDS\PAGE. This phenomenon of slower migration has been observed with other MutT motif proteins, including human Ap % A hydrolase [6] and S. cere isiae Ap ' A hydrolase [18] and might result from incomplete denaturation and SDS binding during sample preparation owing to the stability imparted by the mixed β-sheet core structure that might be a feature of this enzyme family [23]. (Figure 6b, lane 3).…”

Functional Significance of the Conserved Residues for the 23-Residue Module among MTH1 and MutT Family Proteins

“…For E. coli MutT, it has been established that the 23-residue sequence constitutes the active center for dGTPase activity (21,32), probably also for the 8-oxo-dGTPase activity, and that the module consists of loop I (Gly 37 -Thr 45 ) and ␣-helix I (Pro 46 -Gly 59 ) (33). The ␣-helix I in MutT is apparently amphipathic, and it was shown that MutT has two hydrophobic cores, one of them consisting of Val , and Lys 39 may be involved in the catalytic reaction (21,22).…”

Structure of a Nudix hydrolase (MutT) in the Mg2+-bound state fromBartonella henselae, the bacterium responsible for cat scratch fever

“…Nudix proteins are all characterized by the presence of a highly conserved 23-residue 'Nudix box', GX 5 EX 7 REUXEEXGU, where U is a bulky hydrophobic group and X is any residue (Abeygunawardana et al, 1995;Bessman et al, 1996). The Nudix sequence adopts a loop-helix-loop motif (Koonin, 1993) that serves to coordinate the catalytically essential divalent cation (usually Mg 2+ ) to the protein (Lin et al, 1997;Bailey et al, 2002;Gabelli et al, 2001).…”

“…1. It is from this general structure, Nucleoside diphosphates linked to some other moiety x, that the acronym 'Nudix' arose (Abeygunawardana et al, 1995;Bessman et al, 1996). Identified Nudix substrates include capped mRNA (Wang, Jiao et al, 2002), dinucleotide coenzymes, nucleotide sugars, nucleotide alcohols, dinucleotide polyphosphates, both canonical and oxidized (deoxy)ribonucleoside triphosphates (NTPs) (Dunn et al, 1999) and canonical (deoxy)ribonucleoside diphosphates (NDPs) (Fisher et al, 2004;Buchko et al, 2008).…”

“…Some Nudix substrates have been reported without a nucleoside, such as thiamine pyrophosphate (Lawhorn et al, 2004), diphosphoinositol polyphosphates (Safrany et al, 1998) and dihydroneopterin triphosphate (Klaus et al, 2005). With a few exceptions, catalysis depends on a highly conserved sequence of amino acids, GX 5 EX 7 REUXEEXGU (where U is an aliphatic and hydrophobic amino acid and X is any amino acid), called the 'Nudix box' (Abeygunawardana et al, 1995;Bessman et al, 1996). This box forms a loop-helix-loop structural motif that is part of the larger substrate-binding Nudix fold.…”