31P NMR and genetic analysis establish hinT as the only Escherchia coli purine nucleoside phosphoramidase and as essential for growth under high salt conditions.
Eukaryotic cells encode AMP-lysine (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester) 5'-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative (31)P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5'-monophosphoramidate (AMP-NH(2)). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 m KCl, 0.9 m NaCl, 0.5 m NaOAc, or 10 mm MnCl(2). Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.
Chou
Tsui-Fen
TF
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Bieganowski
Pawel
P
Shilinski
Kara
K
Cheng
Jilin
J
Brenner
Charles
C
Wagner
Carston R
CR
D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D049109:Cell Proliferation; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D006151:Guanosine / Q000737:chemistry; D006157:Guanosine Monophosphate / Q000737:chemistry; D006801:Humans; D006867:Hydrolases / Q000737:chemistry; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011684:Purine Nucleosides / Q000737:chemistry; D011687:Purines / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012492:Salts / Q000494:pharmacology*; D013379:Substrate Specificity; D013997:Time Factors; D019076:Transgenes
D011684:Purine Nucleosides; D011687:Purines; D011994:Recombinant Proteins; D012492:Salts; C097552:histidine triad protein; D006151:Guanosine; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D006867:Hydrolases; D008239:Lysine; C030985:purine