FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1513015502> ?p ?o ?g. }

• W1513015502 endingPage "1534" @default.
• W1513015502 startingPage "1529" @default.
• W1513015502 abstract "An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (Mr = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 μmol min−1 mg−1 mM−1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Kmfor NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5′-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 μM. An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (Mr = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 μmol min−1 mg−1 mM−1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Kmfor NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5′-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 μM. This work was initiated during a study of a highly conserved motif that was first identified in the Escherichia coli MutT and Streptococcus pneumoniae MutX antimutator proteins by Mejean et al.(1Mejean V. Salles C. Bullions L.C. Bessman M.J. Claverys J.P. Mol. Microbiol. 1994; 11: 323-330Crossref PubMed Scopus (54) Google Scholar). The motif is shared by enzymes from Proteus vulgaris(2Kamath A.V. Yanofsky C. Gene (Amst.). 1993; 134: 99-102Crossref PubMed Scopus (35) Google Scholar), human cells(3Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar), and another partially purified E. coli protein(4Bullions L.C. Mejean V. Claverys J.P. Bessman M.J. J. Biol. Chem. 1994; 269: 12339-12344Abstract Full Text PDF PubMed Google Scholar). Because these proteins all hydrolyze nucleoside triphosphates to form nucleoside monophosphates and inorganic pyrophosphate, we proposed that this small region of conserved amino acids, common to these proteins, designates a nucleoside-triphosphate pyrophosphohydrolase activity(4Bullions L.C. Mejean V. Claverys J.P. Bessman M.J. J. Biol. Chem. 1994; 269: 12339-12344Abstract Full Text PDF PubMed Google Scholar). Several other open reading frames in widely divergent organisms that specify this small sequence of amino acids but that otherwise have little homology were identified by a computer search of the data bases(1Mejean V. Salles C. Bullions L.C. Bessman M.J. Claverys J.P. Mol. Microbiol. 1994; 11: 323-330Crossref PubMed Scopus (54) Google Scholar, 5Koonin E.V. Nucleic Acids Res. 1993; 21: 4847Crossref PubMed Scopus (98) Google Scholar). We have initiated a systematic study to isolate and examine whether these other genes code for proteins that share similar enzymatic activities. One of these proteins, coded for by open reading frame orf257 (or yjad) between the thiC and hemeE genes in the 90-min region of the E. coli genetic map (6Kano Y. Osato K. Wada M. Imamoto F. Mol. & Gen. Genet. 1987; 209: 408-410Crossref PubMed Scopus (72) Google Scholar) (GenBank™ accession numbers U00006 and D12624), is the subject of this study. It will be demonstrated that this protein does not hydrolyze nucleoside triphosphates, but that it has a novel NADH pyrophosphatase activity, which provides further insight into the function of the conserved amino acid motif. Primers were obtained from Integrated DNA Technologies (Coralville, Iowa). Unless otherwise noted, biochemicals and enzymes were purchased from Sigma. E. coli strain MG1655, kindly provided by Dr. Frederick R. Blattner, was used to prepare chromosomal DNA by the method of Silhavy et al.(7Silhavy T.J. Berman M.L. Enquist L.W. DNA Extractions from Bacterial Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984Google Scholar). Oligonucleotide primers were used in the polymerase chain reaction to attach NcoI and BamHI restriction sites and to amplify the orf257 gene. The 790-base pair product was purified from an agarose gel, digested with NcoI and BamHI (Stratagene, La Jolla, CA), repurified, and ligated into the NcoI and BamHI sites of plasmid pET11d (Novagen, Madison, WI). In the resulting plasmid, the orf257 gene was under control of a T7(lac) promoter. The plasmid, pETorf257, was transformed into strain HB101 for storage and into strain HMS174(DE3) for protein expression using techniques from Sambrook et al.(8Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Strain HMS174(DE3), harboring plasmid pETorf257, was unstable when grown to high cell density, presumably due to a selective disadvantage upon depletion of ampicillin. Consequently, cultures were not grown directly to stationary phase, and the following procedure was designed to maximize the yield of the Orf257 protein. Single colonies of strain HMS174(DE3) containing pETorf257 were inoculated into 5 ml of LB medium containing 100 μg/ml ampicillin. After the cells grew to an A600 of 0.5, they were collected by centrifugation, washed with 0.9% NaCl, and transferred to 100 ml of fresh medium containing ampicillin. This wash was repeated after the cells again reached an A600 of 0.5, and the cells were then transferred to 2 liters of fresh medium. When the cells again reached an A600 of 0.5, they were induced with 1 mM isopropyl-β-D-thiogalactopyranoside and grown for an additional 2 h. Under these conditions, ∼100% of the cells still contained the plasmid, as determined by plating aliquots of the culture on selective media. The induced cells were harvested by centrifugation and washed with an isotonic saline solution, and the cell paste was suspended in buffer A (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, and 1 mM dithiothreitol) and sonicated in a Branson sonifier for 20 min. The crude extract was clarified by centrifugation at 15,000 × g for 30 min (Fraction I). Fraction I was diluted to 10 mg/ml, and a 10% solution of streptomycin sulfate was added slowly to a final concentration of 1% while the extract was stirred on ice. The precipitate, which contained the Orf257 protein, was collected by centrifugation and dissolved in 20 ml of buffer A (Fraction II). Fraction II (18 ml) was loaded onto a column (2.5 × 24 cm) of DEAE-Sepharose (Pharmacia Biotech Inc.) and washed with 400 ml of 50 mM Tris-Cl, pH 7.5, 0.1 mM dithiothreitol (buffer B). The Orf257 protein was eluted with buffer B containing 200 mM NaCl, whereas nucleic acids remained bound to this column. The fractions containing the Orf257 protein were pooled (173 ml), and EDTA was added to a final concentration of 1 mM (Fraction III). Solid ammonium sulfate (50 g) was added to 172 ml of Fraction III, and the precipitate was collected by centrifugation and discarded. Ammonium sulfate (53.6 g) was added to the supernatant, and the precipitate containing the Orf257 protein was dissolved in 7.5 ml of buffer A (Fraction IV). Fraction IV (7 ml) was loaded onto a gel filtration column (Sephadex G-100, 2.5 × 60 cm) and eluted with 50 mM Tris-Cl, 1 mM EDTA, 0.1 mM dithiothreitol, and 50 mM NaCl. The fractions containing the Orf257 protein were combined, concentrated by ultrafiltration, dialyzed against buffer A, and stored at −80°C in 30% glycerol (Fraction V). The purification of the enzyme was originally followed by polyacrylamide gel electrophoresis (9Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (204445) Google Scholar) because we had not yet discovered an activity associated with the Orf257 protein. Once the major substrate of the enzyme was discovered, the activity was quantitated by three assays, all based on the measurement of the AMP formed in. Mg2+NADH⟶Orf257NMNH+AMP The reaction mixtures (100 μl for Assays 1 and 2 and 50 μl for Assay 3) contained 50 mM Tris-Cl, pH 8.5, 20 mM MgCl2, and 0.33-3.3 milliunits of enzyme. One unit of activity is 1 μmol of NADH hydrolyzed per min. After incubation for 10-30 min at 37°C, the reactions were terminated by boiling. Stage I was then treated as follows. The AMP formed was measured by converting it to ADP with adenylate kinase and ATP and coupling this to the lactate dehydrogenase indicator system, which follows the oxidation of NADH spectrally at 340 nm(10Maley F. Ochoa S. J. Biol. Chem. 1958; 233: 1538-1543Abstract Full Text PDF PubMed Google Scholar). The reaction contained 0.1 ml of Stage I, 20 mM KCl, 6 mM ATP, 10 mM MgCl2, 4 mM phosphoenolpyruvate, 0.4 mM NADH, 10 units of lactate dehydrogenase, 10 units of pyruvate kinase, and 5 units of adenylate kinase in a volume of 1 ml. Calculation was based on a value of 6.22 × 103M−1 cm−1 for ∊340 NADH. The AMP was measured by its spectral shift at 265 nm when hydrolyzed to IMP with AMP deaminase (11Nikiforuk G. Colowick S.P. Methods Enzymol. 1955; 2: 469-473Crossref Scopus (18) Google Scholar) based on the method developed by Kalckar(12Kalckar H.M. J. Biol. Chem. 1947; 176: 461-475Abstract Full Text PDF Google Scholar). In addition to Stage I, the reaction contained 160 mM sodium succinate, pH 6.0, 10 mM MgCl2, and 0.25 unit of AMP deaminase in a volume of 1 ml. The AMP and NMNH 1The abbreviations used are: NMNHreduced nicotinamide mononucleotideAppAdi(adenosine 5′)-P1,P2-pyrophosphateApppAdi(adenosine 5′)-P1,P3-triphosphateAppppAdi(adenosine 5′)-P1,P4-tetraphosphate. formed in Stage I were hydrolyzed to the respective nucleosides and inorganic orthophosphate, and the latter was determined colorimetrically. To Stage I, 1-2 units of calf intestinal alkaline phosphatase (EC 3.1.3.1) or 5′-nucleotidase (EC 3.1.3.5) were added and incubated for an additional 30 min at 37°C. Phosphate was determined by the method of Ames and Dubin (13Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar) as modified by Bhatnagar et al.(14Bhatnagar S.K. Bullions L.C. Bessman M.J. J. Biol. Chem. 1991; 266: 9050-9054Abstract Full Text PDF PubMed Google Scholar). reduced nicotinamide mononucleotide di(adenosine 5′)-P1,P2-pyrophosphate di(adenosine 5′)-P1,P3-triphosphate di(adenosine 5′)-P1,P4-tetraphosphate. The orf257 gene was cloned directly from chromosomal DNA isolated from strain MG1655 as described under “Methods.” The DNA was inserted into the vector pET11d, and the region containing the insert was sequenced using standard procedures (Sequenase Version 2.0). The nucleotide sequence agreed with that submitted to GenBank™ (accession number U00006) and designated a protein containing 257 amino acids as shown in Fig. 1. The region of homology to the MutT protein and other MutT-like proteins spans amino acids 159-181, and all of the amino acids in the signature sequence identified by Koonin (5Koonin E.V. Nucleic Acids Res. 1993; 21: 4847Crossref PubMed Scopus (98) Google Scholar) are also present in this region of the Orf257 protein. These amino acids were used as the basis for the alignment with the MutT protein. Cultures of HMS174(DE3) cells containing pETorf257 were grown, harvested, extracted, and fractionated as described under “Methods.” The overexpression of the Orf257 protein in this system is shown in Fig. 2A by the appearance of a new band at ∼29 kDa after induction of the cells with isopropyl-β-D-thiogalactopyranoside. Also shown is an aliquot of the final stage in the purification, which appears >98% pure. The protein appears virtually free of contaminants on a native gel as well (Fig. 2B), and assays of slices from this gel indicated that the enzymatic activity coincided with the major protein band (data not shown). It was noted that the enzyme precipitates with the nucleic acids during the streptomycin fractionation. This may indicate that the enzyme binds to DNA or RNA, although no subsequent evidence of nucleic acid involvement such as stimulation or inhibition of activity was observed in the presence of single- or double-stranded DNA or RNA. The protein eluted from the gel filtration column in Fraction V earlier than would be expected for a 29-kDa protein. A second gel filtration column was calibrated with molecular weight standards, and the Orf257 protein appeared at a region of ∼60 kDa. This could mean that Orf257 exists as a homodimer in solution, although further physicochemical techniques would be necessary to substantiate this possibility. We initially thought that the Orf257 protein would have a nucleoside-triphosphate pyrophosphohydrolase activity similar to that of all other members of the MutT family of proteins that have been characterized(3Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar, 4Bullions L.C. Mejean V. Claverys J.P. Bessman M.J. J. Biol. Chem. 1994; 269: 12339-12344Abstract Full Text PDF PubMed Google Scholar, 15Bhatnagar S.K. Bessman M.J. J. Biol. Chem. 1988; 263: 8953-8957Abstract Full Text PDF PubMed Google Scholar). No such activity was detected for the Orf257 protein using the eight canonical (deoxy)nucleoside triphosphates as substrates. To test the possibility that the protein was missing a pyrophosphate acceptor or a cofactor, we set up a coupled lactate dehydrogenase/pyruvate kinase/adenylate kinase assay to test for a substrate-dependent formation of ADP or AMP by monitoring NADH oxidation at 340 nm. All fractions containing the Orf257 protein were found to contain an adenylate kinase-dependent (AMP-producing) activity, and furthermore, this activity was later observed even when ATP was absent from the assay mixture. The only other direct source of AMP in the system was NADH. Accordingly, a reaction mixture containing only NADH, MgCl2, and the Orf257 protein was analyzed by paper electrophoresis in 25 mM citrate at pH 4.9 as described by Markham and Smith(16Markham R. Smith J.D. Biochem. J. 1952; 52: 552-557Crossref PubMed Scopus (172) Google Scholar). The products, visualized with UV light, comigrated with authentic AMP and NMNH, respectively. The AMP product was confirmed by its reactivity with adenylate deaminase, which is highly specific for this nucleotide(11Nikiforuk G. Colowick S.P. Methods Enzymol. 1955; 2: 469-473Crossref Scopus (18) Google Scholar). The adenylate deaminase assay and the phosphate assay described under “Methods” were used to measure the stoichiometry of the reaction as shown in Fig. 3. No phosphate was released upon digestion of NADH with the Orf257 protein alone. Upon subsequent addition of alkaline phosphatase, 2 mol of phosphate were detected for each mole of AMP formed (with the second mole of Pi coming from NMNH). The pH optimum for the reaction was 8.5 (data not shown). Both MutT (4Bullions L.C. Mejean V. Claverys J.P. Bessman M.J. J. Biol. Chem. 1994; 269: 12339-12344Abstract Full Text PDF PubMed Google Scholar) and the MutT-like enzyme Orf17 2D. N. Frick and M. J. Bessman, unpublished results. also have alkaline pH optima. Although phosphate buffer inhibited the reaction slightly (20%), dithiothreitol, DNA, monovalent cations, pyrophosphate, and EDTA (at concentrations significantly lower than metal concentration) had no effect on activity. The lack of inhibition or stimulation by pyrophosphate clearly distinguished the enzyme from the reversible NAD+ pyrophosphorylase (EC 2.7.7.1)(17Kornberg A. J. Biol. Chem. 1950; 182: 779-793Abstract Full Text PDF Google Scholar). Only weak product inhibition was observed for NMNH, which indicates that the reaction is not freely reversible. A double reciprocal analysis of four titrations with NADH (0.2-1.0 mM) in the presence of various concentrations of NMNH (0-2.1 mM) suggested that NMNH is a linear competitive inhibitor, with a Ki of 4.2 mM. No inhibition was observed for the oxidized NMN+ even at 10-fold higher inhibitor concentrations (data not shown). Several nucleotide pyrophosphates were tested as substrates, and their relative activities are shown in Table 1. All dinucleotides were β-isomers because the α-isomer of NAD+ was not a substrate for the enzyme. Reactions were done at 37°C in 50 mM Tris-Cl, 20 mM MgCl2, 2 mM each substrate, and 0.2-20 μg (1.5-150 milliunits) of the Orf257 protein. Because initial velocity is approximately equal to the Vmax for NADH under these conditions, the values reported represent the specific activities of each substrate relative to that of NADH. The data in Table 1 were obtained using Assay 3 (phosphatase-labile Pi). In each case where AMP was a potential product, Assay 2 was used also, as confirmatory evidence, with essentially identical results (data not shown).Tabled 1 Open table in a new tab The enzyme was more active on NADH than on any other substrate tested, and in each case involving the pyridine nucleotides, the reduced substrate was hydrolyzed more rapidly than its respective oxidized form. Several other nucleotides with related structural features showed varying degrees of reactivity. However, it is especially noteworthy that none of the 16 canonical (deoxy)nucleoside di- and triphosphates were hydrolyzed. This is in striking contrast to the other enzymes of the family (MutT, MutX, Orf17, and human 8-oxo-dGTPase), which all show a marked preference for deoxynucleoside triphosphates. Certain patterns emerge in comparing the reactivity of several of the other substrates. For example, the rate of hydrolysis decreases as the length of the polyphosphate bridge increases in the ApnA series. Furthermore, a second nucleotide is essential for optimal activity. The elimination of the nicotinamide group (ADP-ribose) causes a 3-fold reduction in rate, and the replacement of the ribose with a glucose (ADP-glucose) causes a further 7-fold decrease. The enzyme has no detectable activity under these conditions toward UDP-glucose in which neither the adenine nor the nicotinamide groups are retained. These observations focus attention on structural features of the substrates that correlate with their reactivity. However, caution should be exercised in overinterpreting the observations made on this small group of compounds examined under one set of reaction conditions. Several of the substrates showing high relative rates (in Table 1) were subjected to a more detailed kinetic analysis. Double reciprocal (Lineweaver-Burk) analysis (18Lineweaver H. Burk D. J. Am. Chem. Soc. 1934; 56: 658-666Crossref Scopus (7793) Google Scholar) and nonlinear least-squares fitting weighted to substrate concentration (19Cleland W.W. Methods Enzymol. 1979; 63: 103-139Crossref PubMed Scopus (1922) Google Scholar) were used to determine the Km and Vmax values of the substrates tested. Table 2 lists the Km, Vmax, and catalytic efficiency (Vmax/Km) of various substrates, which again demonstrate that NADH is the most favored substrate for the Orf257 protein identified so far. The replacement of the adenine moiety with hypoxanthine affects the Km or Vmax slightly (<3-fold). However, changes on the nicotinamide nucleotide have much larger effects on both the Km and Vmax. The substitution of the nicotinamide group with adenine (AppA) or the elimination of the base entirely (ADP-ribose) causes an order of magnitude decrease in Vmax/Km, and the introduction of a positive charge on the nicotinamide is associated with a decrease in Vmax/Km by over 2 orders of magnitude.Tabled 1 Open table in a new tab The striking preference for NADH over NAD+, which may be related to the physiological function of the Orf257 protein, is clearly demonstrated in Fig. 4. For NADH, the Vmax (7.6 units mg−1) corresponds to a turnover rate of 3.8/s based on two active sites/dimer. This is similar to the kcat for the MutT enzyme(14Bhatnagar S.K. Bullions L.C. Bessman M.J. J. Biol. Chem. 1991; 266: 9050-9054Abstract Full Text PDF PubMed Google Scholar, 20Frick D.N. Weber D.J. Gillespie J.R. Bessman M.J. Mildvan A.S. J. Biol. Chem. 1994; 269: 1794-1803Abstract Full Text PDF PubMed Google Scholar). The AMP deaminase assay (Assay 2) was used to explore the metal ion requirement of the enzyme. The chlorides of Mg2+, Mn2+, Zn2+, Co2+, Fe2+, Cu2+, and Ca2+ at concentrations from 0.01 to 10 mM were compared, and at optimal concentrations of each metal, Zn2+, Co2+, and Fe2+ gave 10, 8, and 8% of the activity of Mg2+, respectively, whereas Cu2+ and Ca2+ supported no detectable hydrolysis. On the other hand, Mn2+, as shown below, supported more than twice the rate of hydrolysis compared with Mg2+ at optimal concentrations. Unlike other dinucleotide pyrophosphatases(21Nakajima Y. Fukunaga N. Sasaki S. Usami S. Biochim. Biophys. Acta. 1973; 293: 242-255Crossref PubMed Scopus (3) Google Scholar), Co2+ was not as effective as Mg2+, and it did not stimulate the enzyme when included with either Mn2+ or Mg2+. Fig. 5 shows a titration of 1 mM NADH with MnCl2 or MgCl2. Double reciprocal analysis (Fig. 5B) yields an apparent Vmax for the Mn2+-activated reaction of 22 units/mg with an apparent Km(Mn2+) of 30 μM and an apparent Vmax for the Mg2+-activated reaction of 8.8 units/mg with an apparent Km(Mg2+) of 3.7 mM. This indicates that at low metal concentrations, Mn2+ has a 310-fold higher Vmax/Km (metal) than Mg2+. However, due to the inhibitory effects of Mn2+, the two metals support roughly equal velocities at high metal concentrations. Since the purification and characterization of NAD+ pyrophosphatase (EC 3.6.1.22) from potato extracts were described by Kornberg and Pricer(22Kornberg A. Pricer W.E. J. Biol. Chem. 1950; 182: 763-778Abstract Full Text PDF Google Scholar), enzymes cleaving NAD+ into NMN+ and AMP have been reported in a large variety of plants, animals, and bacteria. The relatedness of these enzymes has been difficult to ascertain because few have been purified, and most have a broad spectrum of activities not only to NAD+ and NADP+, but also to FAD and ADP-ribose and, in some cases, to ATP, ADP, and the family of bis(5′-nucleosidyl) oligophosphates as represented by, for example, diadenosine tetraphosphate (AppppA). However, only one other enzymatic activity has been reported that cleaves NADH at a higher rate than NAD+, as is the case with the enzyme described in this paper. Jacobson and Kaplan (23Jacobson K.B. Kaplan N.O. J. Biol. Chem. 1957; 226: 427-437Abstract Full Text PDF PubMed Google Scholar) partially purified a dinucleotide pyrophosphatase from pigeon liver extracts that hydrolyzed NADH at a higher rate than NAD+, but their enzyme is clearly distinguished from the E. coli enzyme by several criteria including its activity on α-NAD+ and its preference for ADP-ribose, which it hydrolyzes at 200% of the rate of NADH. The E. coli enzyme is not active on α-NAD+, and its Vmax/Km for NADH is 20-fold higher than for ADP-ribose. Further comparisons of the E. coli enzyme described in this paper with previously reported related activities is complicated by the fact that the earlier work was done prior to the development of techniques for gene analysis. Of what use to the cell is an enzyme that hydrolyzes and essentially inactivates an important cofactor in cellular metabolism? One possibility is that NMNH plays some undiscovered role in the cell. The only way known, so far, of generating NMNH is through the hydrolysis of NADH by this enzyme. A second, more general role of the enzyme could be the regulation of the intracellular NADH/NAD+ ratio, which is known to be an important factor in maintaining a balance between anabolic and catabolic pathways in higher organisms. Oxidoreduction reactions in which NAD+ and NADH participate as cofactors are, in most cases, freely reversible, and the selective removal of NADH would favor the oxidative pathway especially under anaerobic conditions, where the reoxidation of NADH would be diminished. It is interesting that Jacobson and Kaplan(23Jacobson K.B. Kaplan N.O. J. Biol. Chem. 1957; 226: 427-437Abstract Full Text PDF PubMed Google Scholar), in their early work, reconstructed a system in which the addition of partially purified pigeon liver NADH pyrophosphatase increased the rate and the extent of formation of acetaldehyde from ethanol by purified alcohol dehydrogenase. Their interpretation was that the selective hydrolysis of NADH shifted the equilibrium in favor of the oxidized substrate. Thus, the NADH pyrophosphatase could substitute for an electron acceptor and influence the equilibrium of a metabolic pathway under a specific set of circumstances. We are constructing a null mutant devoid of the enzyme in order to uncover its functional significance. Clinical interest has been generated by the report (24Yamashina I. Yoshida H. Fukui S. Funakoshi I. Mol. Cell. Biochem. 1983; 52: 107-124Crossref PubMed Scopus (23) Google Scholar, 25Yoshida H. Fukui S. Funakoshi I. Yamashina I. J. Biochem. (Tokyo). 1983; 93: 1641-1648Crossref PubMed Scopus (13) Google Scholar) that Lowe's syndrome, a genetic disease with pleiotropic sequelae, is tied to an overproduction of a dinucleotide pyrophosphatase. Our major interest in the enzyme is that it shares a signature sequence, GXU(X)3ET(X)6REUXEE (where U represents the bulky aliphatic residue L, I, or V), with four other proteins that we have partially characterized and that all have nucleoside-triphosphate hydrolase activities producing nucleoside monophosphates and inorganic pyrophosphate(1Mejean V. Salles C. Bullions L.C. Bessman M.J. Claverys J.P. Mol. Microbiol. 1994; 11: 323-330Crossref PubMed Scopus (54) Google Scholar, 4Bullions L.C. Mejean V. Claverys J.P. Bessman M.J. J. Biol. Chem. 1994; 269: 12339-12344Abstract Full Text PDF PubMed Google Scholar, 15Bhatnagar S.K. Bessman M.J. J. Biol. Chem. 1988; 263: 8953-8957Abstract Full Text PDF PubMed Google Scholar). Sakumi et al.(3Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar) have described an 8-oxo-dGTPase activity from human cells that also has the same amino acid motif. Besides this small region of identity, the six proteins (including the enzyme described in this paper) share very little similarity. Our analyses of site-directed mutations in MutT implicate this small region, common to all the proteins, as being important in the enzymatic activity of the enzymes (1Mejean V. Salles C. Bullions L.C. Bessman M.J. Claverys J.P. Mol. Microbiol. 1994; 11: 323-330Crossref PubMed Scopus (54) Google Scholar, 26Frick D.N. Bullions L.C. Townsend B.D. Bessman M.J. FASEB J. 1994; 8 (abstr.): A1278Google Scholar), and NMR structural analysis of MutT has revealed that this region of the protein is involved in nucleotide binding(27Weber D.J. Abeygunawardana C. Bessman M.J. Mildvan A.S. Biochemistry. 1993; 32: 13081-13088Crossref PubMed Scopus (37) Google Scholar). Four of the six proteins from E. coli, S. pneumoniae, P. vulgaris, and human cells (1Mejean V. Salles C. Bullions L.C. Bessman M.J. Claverys J.P. Mol. Microbiol. 1994; 11: 323-330Crossref PubMed Scopus (54) Google Scholar, 2Kamath A.V. Yanofsky C. Gene (Amst.). 1993; 134: 99-102Crossref PubMed Scopus (35) Google Scholar, 3Sakumi K. Furuichi M. Tsuzuki T. Kakuma T. Kawabata S. Maki H. Sekiguchi M. J. Biol. Chem. 1993; 268: 23524-23530Abstract Full Text PDF PubMed Google Scholar, 15Bhatnagar S.K. Bessman M.J. J. Biol. Chem. 1988; 263: 8953-8957Abstract Full Text PDF PubMed Google Scholar) prevent mutations caused by AT → CG transversions. A fifth protein, coded for by E. coli orf17, has not, as yet, been shown to prevent mutations, but it is a nucleoside triphosphatase with a preference for dATP(4Bullions L.C. Mejean V. Claverys J.P. Bessman M.J. J. Biol. Chem. 1994; 269: 12339-12344Abstract Full Text PDF PubMed Google Scholar). These five proteins, which only have this small signature sequence in common, catalyze the hydrolysis of nucleoside triphosphates according to the following scheme: (deoxy)nucleoside triphosphate → (deoxy)nucleoside monophosphate + PPi. Until we discovered the NADH pyrophosphatase described in this report, we believed that the signature sequence designated a catalytic site specific for the attack on the beta-phosphate of a nucleoside triphosphate with the elimination of pyrophosphate(28Weber D.J. Bhatnagar S.K. Bullions L.C. Bessman M.J. Mildvan A.S. J. Biol. Chem. 1992; 267: 16939-16942Abstract Full Text PDF PubMed Google Scholar). Because NADH pyrophosphatase catalyzes the cleavage of NADH → NMNH + AMP, we believe that this signature sequence, which in the MutT protein forms a loop-helix-loop motif (27Weber D.J. Abeygunawardana C. Bessman M.J. Mildvan A.S. Biochemistry. 1993; 32: 13081-13088Crossref PubMed Scopus (37) Google Scholar) not seen in other nucleotide-binding sites(29Schultz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Crossref Scopus (299) Google Scholar), has been conserved during evolution and adapted to participate in different metabolic reactions involving the cleavage of a nucleotide pyrophosphate bond. Computer searches of the sequence data banks (1Mejean V. Salles C. Bullions L.C. Bessman M.J. Claverys J.P. Mol. Microbiol. 1994; 11: 323-330Crossref PubMed Scopus (54) Google Scholar, 5Koonin E.V. Nucleic Acids Res. 1993; 21: 4847Crossref PubMed Scopus (98) Google Scholar) have revealed several other genes of unknown function specifying the same amino acid motif in a variety of organisms ranging from viruses to eucaryotes. We are in the process of cloning, expressing, and identifying enzyme activities associated with these genes. We are also determining the three-dimensional solution structure (27Weber D.J. Abeygunawardana C. Bessman M.J. Mildvan A.S. Biochemistry. 1993; 32: 13081-13088Crossref PubMed Scopus (37) Google Scholar) and crystal structure 3S. Quirk and M. J. Bessman, unpublished results. of some of these enzymes. These approaches should lead to an understanding of the basic biochemistry associated with the conserved amino acid motif and perhaps identify functions for the other genes containing sequences that specify this arrangement of amino acids. We thank Dr. Frederick R. Blattner for the gift of strain MG1655 and Dorothy Regula for help in the preparation of the manuscript." @default.
• W1513015502 created "2016-06-24" @default.
• W1513015502 creator A5060924856 @default.
• W1513015502 creator A5079193494 @default.
• W1513015502 date "1995-01-01" @default.
• W1513015502 modified "2023-10-17" @default.
• W1513015502 title "Cloning, Purification, and Properties of a Novel NADH Pyrophosphatase" @default.
• W1513015502 cites W1512546645 @default.
• W1513015502 cites W1538191341 @default.
• W1513015502 cites W1541512177 @default.
• W1513015502 cites W1558506222 @default.
• W1513015502 cites W1594443532 @default.
• W1513015502 cites W1635858559 @default.
• W1513015502 cites W1658844382 @default.
• W1513015502 cites W1981962835 @default.
• W1513015502 cites W1992246409 @default.
• W1513015502 cites W2040739506 @default.
• W1513015502 cites W204889168 @default.
• W1513015502 cites W2054979333 @default.
• W1513015502 cites W2062006489 @default.
• W1513015502 cites W2067732613 @default.
• W1513015502 cites W2079079715 @default.
• W1513015502 cites W2100837269 @default.
• W1513015502 cites W2108902713 @default.
• W1513015502 cites W2136967256 @default.
• W1513015502 cites W2217234049 @default.
• W1513015502 cites W2256273377 @default.
• W1513015502 cites W25726284 @default.
• W1513015502 cites W2969273703 @default.
• W1513015502 cites W4236230108 @default.
• W1513015502 cites W61410340 @default.
• W1513015502 cites W6204808 @default.
• W1513015502 cites W73321846 @default.
• W1513015502 doi "https://doi.org/10.1074/jbc.270.4.1529" @default.
• W1513015502 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7829480" @default.
• W1513015502 hasPublicationYear "1995" @default.
• W1513015502 type Work @default.
• W1513015502 sameAs 1513015502 @default.
• W1513015502 citedByCount "93" @default.
• W1513015502 countsByYear W15130155022012 @default.
• W1513015502 countsByYear W15130155022013 @default.
• W1513015502 countsByYear W15130155022014 @default.
• W1513015502 countsByYear W15130155022015 @default.
• W1513015502 countsByYear W15130155022016 @default.
• W1513015502 countsByYear W15130155022017 @default.
• W1513015502 countsByYear W15130155022018 @default.
• W1513015502 countsByYear W15130155022019 @default.
• W1513015502 countsByYear W15130155022020 @default.
• W1513015502 countsByYear W15130155022021 @default.
• W1513015502 countsByYear W15130155022022 @default.
• W1513015502 countsByYear W15130155022023 @default.
• W1513015502 crossrefType "journal-article" @default.
• W1513015502 hasAuthorship W1513015502A5060924856 @default.
• W1513015502 hasAuthorship W1513015502A5079193494 @default.
• W1513015502 hasBestOaLocation W15130155021 @default.
• W1513015502 hasConcept C121050878 @default.
• W1513015502 hasConcept C181199279 @default.
• W1513015502 hasConcept C185592680 @default.
• W1513015502 hasConcept C199360897 @default.
• W1513015502 hasConcept C2776043855 @default.
• W1513015502 hasConcept C2777008805 @default.
• W1513015502 hasConcept C2780091537 @default.
• W1513015502 hasConcept C41008148 @default.
• W1513015502 hasConcept C55493867 @default.
• W1513015502 hasConceptScore W1513015502C121050878 @default.
• W1513015502 hasConceptScore W1513015502C181199279 @default.
• W1513015502 hasConceptScore W1513015502C185592680 @default.
• W1513015502 hasConceptScore W1513015502C199360897 @default.
• W1513015502 hasConceptScore W1513015502C2776043855 @default.
• W1513015502 hasConceptScore W1513015502C2777008805 @default.
• W1513015502 hasConceptScore W1513015502C2780091537 @default.
• W1513015502 hasConceptScore W1513015502C41008148 @default.
• W1513015502 hasConceptScore W1513015502C55493867 @default.
• W1513015502 hasIssue "4" @default.
• W1513015502 hasLocation W15130155021 @default.
• W1513015502 hasOpenAccess W1513015502 @default.
• W1513015502 hasPrimaryLocation W15130155021 @default.
• W1513015502 hasRelatedWork W1508569549 @default.
• W1513015502 hasRelatedWork W2005243174 @default.
• W1513015502 hasRelatedWork W2020574343 @default.
• W1513015502 hasRelatedWork W2033976912 @default.
• W1513015502 hasRelatedWork W2056014377 @default.
• W1513015502 hasRelatedWork W2057663365 @default.
• W1513015502 hasRelatedWork W2079583561 @default.
• W1513015502 hasRelatedWork W2111716244 @default.
• W1513015502 hasRelatedWork W2128339794 @default.
• W1513015502 hasRelatedWork W2748352372 @default.
• W1513015502 hasVolume "270" @default.
• W1513015502 isParatext "false" @default.
• W1513015502 isRetracted "false" @default.
• W1513015502 magId "1513015502" @default.
• W1513015502 workType "article" @default.