Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis.
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.
Gabelli
Sandra B
SB
Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Bianchet
Mario A
MA
Xu
WenLian
W
Dunn
Christopher A
CA
Niu
Zhi-Dian
ZD
Amzel
L Mario
LM
Bessman
Maurice J
MJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005492:Folic Acid / Q000032:analysis / Q000096:biosynthesis*; D005798:Genes, Bacterial; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019798:Neopterin / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014961:X-Ray Diffraction; D000096964:Nudix Hydrolases
D003838:Deoxyadenine Nucleotides; D005492:Folic Acid; D019798:Neopterin; D011755:Pyrophosphatases; D000096964:Nudix Hydrolases; C013490:dihydroneopterin triphosphate; C026600:2'-deoxyadenosine triphosphate