General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG.
To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.
Banting and Best Department of Medical Research, University of Toronto, Ontario M5G 1L6, Canada.
Michael
M
Proudfoot
Ekaterina
E
Kuznetsova
Greg
G
Brown
Narayana N
NN
Rao
Masanari
M
Kitagawa
Hirotada
H
Mori
Alexei
A
Savchenko
Alexander F
AF
Yakunin
D000135:Acid Phosphatase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D009708:Nucleotidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D003854:Deoxyribonucleotides; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009711:Nucleotides; D011122:Polyphosphates; D012265:Ribonucleotides; D009708:Nucleotidases; C497358:YfbR protein, E coli; C497359:YjjG protein, E coli; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; C089634:umpG protein, E coli; D009699:N-Glycosyl Hydrolases