31P NMR and genetic analysis establish hinT as the only Escherchia coli purine nucleoside phosphoramidase and as essential for growth under high salt conditions

Tsui Fen Chou, Pawel Bieganowski, Kara Shilinski, Juin Cheng, Charles Brenner, Carston R Wagner

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Abstract

Eukaryotic cells encode AMP-lysine (AMP-N-ε-(N-α-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 31P 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-NH2). 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 MnCl2. Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.

Original languageEnglish (US)
Pages (from-to)15356-15361
Number of pages6
JournalJournal of Biological Chemistry
Volume280
Issue number15
DOIs
StatePublished - Apr 15 2005

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Purine Nucleosides
Adenosine Monophosphate
Salts
Escherichia coli
Nuclear magnetic resonance
Adenosine
Substrates
Growth
Lysine
Rabbits
Guanosine
Eukaryotic Cells
Hydrolases
Enzymes
Histidine
Cations
Cell growth
Carrier Proteins
Esters
Hydrolysis

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31P NMR and genetic analysis establish hinT as the only Escherchia coli purine nucleoside phosphoramidase and as essential for growth under high salt conditions. / Chou, Tsui Fen; Bieganowski, Pawel; Shilinski, Kara; Cheng, Juin; Brenner, Charles; Wagner, Carston R.

In: Journal of Biological Chemistry, Vol. 280, No. 15, 15.04.2005, p. 15356-15361.

Research output: Contribution to journalArticle

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AU - Bieganowski, Pawel

AU - Shilinski, Kara

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AU - Brenner, Charles

AU - Wagner, Carston R

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