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; Carston R Wagner

doi:10.1074/jbc.M500434200

Back

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

Journal article

Open access

Peer reviewed

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

The Journal of biological chemistry, Vol.280(15), pp.15356-15361

04/15/2005

DOI: 10.1074/jbc.M500434200

PMCID: PMC2556068

PMID: 15703176

Files and links (1)

url

https://doi.org/10.1074/jbc.M500434200View

Published (Version of record) Open Access

Abstract

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.

Guanosine Monophosphate - chemistry

Rabbits

Cell Proliferation

Escherichia coli - enzymology

Magnetic Resonance Spectroscopy - methods

Models, Chemical

Purines - metabolism

Humans

Adenosine Monophosphate - chemistry

Purine Nucleosides - chemistry

Recombinant Proteins - chemistry

Substrate Specificity

Plasmids - metabolism

Hydrolysis

Salts - pharmacology

Animals

Guanosine - chemistry

Time Factors

Escherichia coli - metabolism

Protein Conformation

Hydrolases - chemistry

Mutation

Lysine - chemistry

Transgenes

Details

Title: Subtitle: 31P NMR and genetic analysis establish hinT as the only Escherchia coli purine nucleoside phosphoramidase and as essential for growth under high salt conditions
Creators: Tsui-Fen Chou - Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA
Pawel Bieganowski
Kara Shilinski
Jilin Cheng
Charles Brenner
Carston R Wagner
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.280(15), pp.15356-15361
DOI: 10.1074/jbc.M500434200
PMID: 15703176
PMCID: PMC2556068
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: R01 CA075954-08 / NCI NIH HHS CA55334 / NCI NIH HHS CA075954 / NCI NIH HHS R01 CA075954 / NCI NIH HHS
Language: English
Date published: 04/15/2005
Academic Unit: Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9983788429502771

Metrics

25 Record Views

50 Times Cited - Web of Science