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J. Biol. Chem., Vol. 279, Issue 31, 32225-32232, July 30, 2004

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Escherichia coli Nucleoside Diphosphate Kinase Interactions with T4 Phage Proteins of Deoxyribonucleotide Synthesis and Possible Regulatory Functions^*

Rongkun Shen, Michael C. Olcott, JuHyun Kim, Indira Rajagopal, and Christopher K. Mathews

From the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7301

In both prokaryotic and eukaryotic organisms, nucleoside diphosphate kinase is a multifunctional protein, with well defined functions in ribo- and deoxyribonucleoside triphosphate biosynthesis and more recently described functions in genetic and metabolic regulation, signal transduction, and DNA repair. This paper concerns two unusual properties of nucleoside diphosphate (NDP) kinase from Escherichia coli: 1) its ability to interact specifically with enzymes encoded by the virulent bacteriophage T4 and 2) its roles in regulating metabolism of the host cell. By means of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays, we have shown that E. coli NDP kinase interacts directly with T4 thymidylate synthase, aerobic ribonucleotide reductase, dCTPase-dUTPase, gene 32 single-strand DNA-binding protein, and deoxycytidylate hydroxymethylase. The interactions with ribonucleotide reductase and with gp32 are enhanced by nucleoside triphosphates, suggesting that the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool. The other investigations in this work stem from the unexpected finding that E. coli NDP kinase is dispensable for successful T4 phage infection, and they deal with two observations suggesting that the NDP kinase protein plays a genetic role in regulating metabolism of the host cell: 1) the elevation of CTP synthetase activity in an ndk mutant, in which the structural gene for NDP kinase is disrupted, and 2) the apparent ability of NDP kinase to suppress anaerobic growth in a pyruvate kinase-negative E. coli mutant. Our data indicate that the regulatory roles are metabolic, not genetic, in nature.

Received for publication, March 11, 2004 , and in revised form, May 26, 2004.

^* This work was supported by National Science Foundation Grant MCB 01 30760. Additional support came from National Institute of Environmental Health Sciences Center Grant ES 00040. 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.

These authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural and Life Sciences Bldg., Corvallis, OR 97331-7301. Tel.: 541-737-1865; Fax: 541-737-1481; E-mail: mathewsc{at}onid.orst.edu.

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				S. C. Goswami, J.-H. Yoon, B. M. Abramczyk, G. P. Pfeifer, and E. H. Postel Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung J. Biol. Chem., October 27, 2006; 281(43): 32131 - 32139. [Abstract] [Full Text] [PDF]

				J. Kim, R. Shen, M. C. Olcott, I. Rajagopal, and C. K. Mathews Adenylate Kinase of Escherichia coli, a Component of the Phage T4 dNTP Synthetase Complex J. Biol. Chem., August 5, 2005; 280(31): 28221 - 28229. [Abstract] [Full Text] [PDF]