Publications

• A nudix enzyme removes pyrophosphate from dihydroneopterin triphosphate in the folate synthesis pathway of bacteria and plants.

  Klaus S.M.J., Wegkamp A., Sybesma W., Hugenholtz J., Gregory J.F. III, Hanson A.D.

  Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of La ... >> More

  Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase. Because many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized. The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a K(m) value of 2 microM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (approximately 1 mM) of Mg(2+), and was active as a monomer. Essentially no reaction occurred without enzyme at 1 mM Mg(2+). Inactivation of ylgG in L. lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis. We therefore propose that ylgG be redesignated as folQ. The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E. coli and shown to have Mg(2+)-dependent DHNTP pyrophosphohydrolase activity. This protein (AtNUDT1) was reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn(2+) (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482-50486). However, we found that this activity is negligible at physiological levels of Mn(2+) and that, with 1 mM Mg(2+), AtNUDT1 prefers DHNTP and (deoxy) nucleoside triphosphates. << Less

  J. Biol. Chem. 280:5274-5280(2005) [PubMed] [EuropePMC]

• The biosynthesis of folic acid. XII. Purification and properties of dihydroneopterin triphosphate pyrophosphohydrolase.

  Suzuki Y., Brown G.M.

  J Biol Chem 249:2405-2410(1974) [PubMed] [EuropePMC]

• Escherichia coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT family of proteins. Cloning, purification, and characterization of the enzyme.

  O'Handley S.F., Frick D.N., Bullions L.C., Mildvan A.S., Bessman M.J.

  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 beta-phosphorus to produce dAMP a ... >> More

  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 beta-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 Mg2+, a Km of 0.8 mM and a kcat of 5.2 s-1 at 37 degrees C for dATP. dAMP is a weak competitive inhibitor with a Ki of approximately 4 mM, while PPi is a much stronger inhibitor with an apparent Ki of approximately 20 microM. The enzyme contains the highly conserved signature sequence GXVEX2ETX6REVXEEX2I 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. << Less

  J. Biol. Chem. 271:24649-24654(1996) [PubMed] [EuropePMC]

• The Lreu_1276 protein from Limosilactobacillus reuteri represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria.

  Kachi K., Sato T., Nagasawa M., Cann I., Atomi H.

  Tetrahydrofolate is a cofactor involved in C₁ metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear ... >> More

  Tetrahydrofolate is a cofactor involved in C₁ metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate. Although two structurally distinct DHNTP pyrophosphohydrolases have been identified in the intestinal bacteria <i>Lactococcus lactis</i> and <i>Escherichia coli</i>, the distribution of their homologs is limited. Here, we aimed to identify a third DHNTP pyrophosphohydrolase gene in the intestinal lactic acid bacterium <i>Limosilactobacillus reuteri</i>. In a gene operon including genes involved in dihydrofolate biosynthesis, we focused on the <i>lreu_1276</i> gene, annotated as Ham1 family protein or XTP/dITP diphosphohydrolase, as a candidate encoding DHNTP pyrophosphohydrolase. The Lreu_1276 recombinant protein was prepared using <i>E. coli</i> and purified. Biochemical analyses of the reaction product revealed that the Lreu_1276 protein displays significant pyrophosphohydrolase activity toward DHNTP. The optimal reaction temperature and pH were 35°C and around 7, respectively. Substrate specificity was relatively strict among 17 tested compounds. Although previously characterized DHNTP pyrophosphohydrolases prefer Mg²⁺, the Lreu_1276 protein exhibited maximum activity in the presence of Mn²⁺, with a specific activity of 28.2 ± 2.0 µmol min^-1 mg^-1 in the presence of 1 mM Mn²⁺. The three DHNTP pyrophosphohydrolases do not share structural similarity to one another, and the distribution of their homologs does not overlap, implying that the Lreu_1276 protein represents a third structurally novel DHNTP pyrophosphohydrolase in bacteria.<h4>Importance</h4>The identification of a structurally novel DHNTP pyrophosphohydrolase in <i>L. reuteri</i> provides valuable information in understanding tetrahydrofolate biosynthesis in bacteria that possess <i>lreu_1276</i> homologs. Interestingly, however, even with the identification of a third family of DHNTP pyrophosphohydrolases, there are still a number of bacteria that do not harbor homologs for any of the three genes while possessing other genes involved in the biosynthesis of the pterin ring structure. This suggests the presence of an unrecognized DHNTP pyrophosphohydrolase gene in bacteria. As humans do not harbor DHNTP pyrophosphohydrolase, the high structural diversity of enzymes responsible for a reaction in tetrahydrofolate biosynthesis may provide an advantage in designing inhibitors targeting a specific group of bacteria in the intestinal microbiota. << Less

  Appl. Environ. Microbiol. 90:e0081424-e0081424(2024) [PubMed] [EuropePMC]

• Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis.

  Gabelli S.B., Bianchet M.A., Xu W., Dunn C.A., Niu Z.-D., Amzel L.M., Bessman M.J.

  Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, ... >> More

  Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 A resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes. << Less

  Structure 15:1014-1022(2007) [PubMed] [EuropePMC]