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,Christopher K Mathews

doi:10.1074/jbc.m402750200

Rongkun Shen, Michael C Olcott + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m402750200

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Abstract

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.

Highlights

Like other large virulent DNA phages, bacteriophage T4 encodes most of the enzymes and proteins needed for its own DNA replication but for biosynthesis of the four deoxyribonucleoside triphosphates
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
Protein-Protein Interactions Involving NDP Kinase—Nucleoside diphosphate kinase occupies a distinctive location on metabolic charts at the interface between nucleotide biosynthesis and nucleotide polymerization, leading both to DNA and RNA

Summary

Introduction

Like other large virulent DNA phages, bacteriophage T4 encodes most of the enzymes and proteins needed for its own DNA replication but for biosynthesis of the four deoxyribonucleoside triphosphates. 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.

Results

Conclusion