A Novel GDP-Mannose Mannosyl Hydrolase Shares Homology with the MutT Family of Enzymes

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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...

Section: Properties Of the Enzymementioning

confidence: 99%

Section: Subcloning Expression and Purificationmentioning

confidence: 99%

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

O'Handley

Frick

Bullions

et al. 1996

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“…Work in progress on the x-ray crystal structure of members of the family should be informative in ascertaining the basis of these observations. 1 Products of the Reaction-With one possible exception (17), virtually all of the Nudix hydrolases studied so far catalyze a nucleophilic attack by water on a pyrophosphate linkage. An analysis of the products of UTP hydrolysis with purified A. tumefaciens UTPase indicates that this enzyme also catalyzes an attack on the pyrophosphate linkage.…”

Section: Resultsmentioning

confidence: 99%

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

Shen

Dunn

et al. 2003

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A new subfamily of the Nudix hydrolases, identified by conserved amino acids upstream and downstream of the Nudix box, has been characterized. The cloned, expressed, and purified orthologous enzymes have major activities on the non-canonical nucleoside triphosphate 5-methyl-UTP (ribo-TTP) and the canonical nucleotide UTP. In addition to their homologous signature sequences and their similar substrate specificities, the members of the subfamily are inhabitants of or are related to the bacterial rhizosphere. We propose the acronym and mnemonic, utp, for the gene designating this unique UTPase.The Nudix hydrolases, so named because they catalyze the hydrolysis of Nucleoside diphosphates linked to some other moiety x (1), constitute a superfamily of enzymes with representatives in all three kingdoms. The members of this superfamily can be identified by a highly conserved amino acid signature sequence called the Nudix box, viz. GXXXXXEXXXXXXXREUXEEXGU SCHEME 1 in which X represents any amino acid and U is a bulky hydrophobic amino acid, usually Ile, Leu, or Val. A current BLAST (2) search of the data banks for polypeptides and expressed sequence tags containing the above sequence, reveals over 1100 open reading frames from more than 250 species ranging from viruses to humans. We have been systematically cloning, expressing, and characterizing members of the superfamily, and without exception, all of the enzymes identified so far hydrolyze nucleoside diphosphate derivatives including nucleotide sugars, dinucleoside polyphosphates, coenzymes, (deoxy)nucleoside triphosphates, and ADP-ribose. Since the same amino acid signature sequence, the Nudix box, represents the nucleotide binding and catalytic site for all these proteins (3-5), the different specificities toward the respective substrates must lie in a region or in regions peripheral to this area. By aligning amino acid sequences of those enzymes hydrolyzing a similar spectrum of substrates, we have identified certain landmark amino acids outside of the Nudix box, enabling us to classify some of the members of the superfamily into distinct subfamilies (6). This has been of singular value, because it has allowed us to predict the enzymatic activity of unidentified open reading frames involved in physiological processes, merely by observing specific telltale amino acid patterns peculiar to each subfamily.In this paper, we describe the cloning, expression, purification, and identification of members of a new subfamily of the Nudix hydrolases, highly active on 5-methyl-UTP (ribo-TTP) and UTP, and recognizable by characteristic amino acid sequences outside of the Nudix box. EXPERIMENTAL PROCEDURES MaterialsThe expression plasmid pET-24a(ϩ) (Km r ) and Escherichia coli HMS174 (DE3) were from Novagen (Madison WI); Agrobacterium tumefaciens strain GV3101 was a gift from Judith Bender, The Johns Hopkins School of Public Health; Pseudomonas aeruginosa, strain BB3/ 216/ATCC27853, was a gift from Thomas Cebula of the Food and Drug Administration; and Caulobacter crescentus stra...

“…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).…”

mentioning

confidence: 99%

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

Dobrzanska¹,

Szurmak²,

Wysłouch‐Cieszyńska³

et al. 2002

The sequence motif commonly called a Nudix box, represented by (GX 5 EX 7 REVXEEXGU) is the marker of a widely distributed family of enzymes that catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we describe the cloning and characterization of an Arabidopsis thaliana cDNA encoding a Nudix hydrolase that degrades NADH. The deduced amino acid sequence of AtNUDT1 contains 147 amino acids. The recombinant AtNUDT1 was expressed in Escherichia coli and purified. In the presence of Mn 2؉ and the optimal pH of 7. 0, the recombinant AtNUDT1 catalyzed the hydrolysis of NADH with a K m value of 0. 36 mM. A V max of 12. 7 units mg ؊1 for NADH was determined. The recombinant AtNUDT1 migrated as a dimer on a gel filtration column. Biochemical analysis of recombinant AtNUDT1 indicated that the first characterized member of the Nudix family from A. thaliana is a NADH pyrophosphatase.