Escherichia coli orf17 Codes for a Nucleoside Triphosphate Pyrophosphohydrolase Member of the MutT Family of Proteins

Abstract

The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the ␤-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg 2؉, a K m of 0.8 mM and a k cat of 5.2 s ؊1 at 37°C for dATP. dAMP is a weak competitive inhibitor with a K i of approximately 4 mM, while PP i is a much stronger inhibitor with an apparent K i of approximately 20 M.The enzyme contains the highly conserved signature sequence GXVEX 2 ETX 6 REVXEEX 2 I designating the MutT family of proteins. However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT ؊ mutator phenotype, and thus must serve a different biological role in the cell.Members of the MutT family of proteins are categorized by a conserved amino acid motif originally identified as an important functional region in the Escherichia coli MutT and the Streptococcus pneumoniae MutX antimutator proteins (1). This conserved amino acid signature sequence was found, by computer search, to be present in a number of open reading frames broadly distributed throughout nature, from viruses to humans (1, 2), and one of these, orf17 (GenBank D10165, 1992), is the subject of this paper. The orf17 gene is located at 41 min on the E. coli chromosome (3) just upstream of the ruvC gene (4, 5), which codes for a Holliday junction-specific endonuclease (4).Both the MutT and MutX proteins are nucleoside triphosphatases (6, 7), as are the corresponding proteins from Proteus vulgaris (8) and from humans (9), and all four are implicated in preventing mutations. On the other hand, two other proteins containing the MutT signature sequence are not nucleoside triphosphatases. One of them, a nucleoside pyrophosphatase, prefers NADH as its substrate (10), while the other, GDPmannose mannosyl hydrolase, prefers GDP-mannose (11). Neither of these latter two enzymes has been linked to a mutagenic pathway. A feature common to all six enzymes containing the signature sequence is the hydrolysis of a substrate containing a nucleoside diphosphate linkage.In this paper, we describe the cloning and expression of the orf17 gene, and the purification and characterization of the Orf17 protein. Like the first four enzymes described above, it is a nucleoside triphosphatase. However, unlike those four enzymes, which prefer dGTP as a substrate over the other canonical (deoxy)nucleoside triphosphates, Orf17 has a unique preference for dATP, and it does not appear to be involved in antimutagenesis. EXERIMENTAL PROCEDURES MaterialsEnzymes-Thermus aquaticus DNA polymerase was from PerkinElmer, and other enzymes used in standard cloning procedures were from Life Technologies, Inc., Stratagene, or U. S. Biochemical Corp. Yeast inorganic pyrophosphata...

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

“…Over 10 putative homologs for bacterial MutT proteins, which also possess the 23-residue sequence and may hydrolyze 8-oxodGTP, can be retrieved by data base searching with the BLASTP alogrithm. In addition to the MTH1 or MutT family proteins, many other proteins with the 23-residue sequence that hydrolyze various nucleotide derivatives and diphosphoinositol derivatives other than 8-oxo-dGTP have been identified (23)(24)(25)(26). Here, we propose that the 23-residue module should be designated as the phosphohydrolase module.…”

“…The 23-residue sequence is a sole conserved sequence among all MutT and MTH1 homologs with 8-oxo-dGTPase, and of the many other proteins with the MutT signature so far identified, some hydrolyze various nucleotide derivatives, such as dATP, diadenosine oligophosphates, NADH, ADP-ribose, and GDPmannose (23)(24)(25). Thus enzymes with the MutT signature are designated as belonging to the Nudix hydrolase family, and the signature is known as Nudix box (18).…”

A New Subfamily of the Nudix Hydrolase Superfamily Active on 5-Methyl-UTP (Ribo-TTP) and UTP

“…Five other Nudix hydrolase nucleoside triphosphatases are known with major activities on dGTP (18), dATP (19), dCTP (15), 3Ј-amino-3Ј-dATP (20), and dTTP 2 all forming the respective monophosphates and inorganic pyrophosphate, classifying them in the category EC 3.6.1.19 with the systematic name nucleoside-triphosphate pyrophosphohydrolase. Thus, the UTPase described herein is properly designated UTP pyrophosphohydrolase, and it is the first of these enzymes preferring ribo-to deoxyribonucleotides.…”

“…The mutation frequencies were calculated from the number of colonies resistant to nalidixic acid or streptomycin as described in O'Handley et al (9).…”

Cloning and Characterization of the First Member of the Nudix Family from Arabidopsis thaliana

“…As all these substrates are either potentially toxic, cell signaling molecules, or metabolic intermediates whose concentrations require modulation during the cell cycle, it has been postulated that the role of Nudix hydrolases is to sanitize or regulate the accumulation of these metabolites (4). To date, a number of Nudix hydrolases from different organisms have been characterized with respect to their major substrates such as nucleotide sugars (5), Ap n A (n ϭ 3, 4, 5, and 6) (6 -12), deoxynucleoside triphosphates (13)(14)(15)(16)(17), ADP-ribose (18 -23), GDP-mannose (5,25), NADH (26 -29), coenzyme A (30 -32), and diphosphoinositol polyphosphates (33)(34)(35).…”