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J. Biol. Chem., Vol. 280, Issue 15, 15356-15361, April 15, 2005

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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^*

Tsui-Fen Chou, Pawel Bieganowski, Kara Shilinski, Jilin Cheng, Charles Brenner, and Carston R. Wagner¶

From the Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455 and the Departments of Genetics and Biochemistry and the Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, New Hampshire 03756

Eukaryotic cells encode AMP-lysine (AMP-N--(N--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.

Received for publication, January 13, 2005

^* This work was supported by U. S. Public Health Service Grant CA55334 (to C. R. W.) and CA075954 (to C. B.) from the National Cancer Institute and a development grant in drug design from the Department of Medicinal Chemistry, University of Minnesota. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains five supplemental figures and two supplemental tables.

^¶ To whom correspondence dhould be addressed: University of Minnesota, Dept. of Medicinal Chemistry, 8-174 Weaver Densford Hall, 308 Harvard St. S.E., Minneapolis, MN 55455. Tel.: 612-625-2614; E-mail: wagne003{at}umn.edu.

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