pyrophosphate and dihydroneopterin-triphosphate

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.

Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Catalysis; Conserved Sequence; Deoxyadenine Nucleotides; Escherichia coli; Folic Acid; Genes, Bacterial; Hydrogen Bonding; Hydrolysis; Kinetics; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutation; Neopterin; Nudix Hydrolases; Open Reading Frames; Plasmids; Protein Binding; Protein Conformation; Protein Structure, Secondary; Pyrophosphatases; Sequence Homology, Amino Acid; Substrate Specificity; X-Ray Diffraction

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.

Topics: Amino Acid Sequence; Diphosphates; Escherichia coli; Folic Acid; Kinetics; Lactococcus lactis; Magnesium; Manganese; Molecular Sequence Data; Neopterin; Nudix Hydrolases; Open Reading Frames; Phylogeny; Plants; Pteridines; Pyrophosphatases; Substrate Specificity