Bacteriophage T4 anaerobic ribonucleotide reductase contains a stable glycyl radical at position 580.
It has been recently recognized that the class III anaerobic ribonucleotide reductase requires the presence of a second activating gene product, NrdG. We have proposed that the role for NrdG involves the generation of an oxygen sensitive glycyl free radical within the NrdD enzyme. In this article we present the generation of such a glycyl free radical within the T4 NrdD subunit and its dependence upon the phage NrdG subunit. Initially, an overexpression system was created that allowed the joint production of T4 NrdD and T4 NrdG. With this system and in the presence of T4 NrdG, an oxygen-sensitive cleavage of NrdD was observed that mimicked the cleavage observed in phage infected Escherichia coli extracts. Under anaerobic conditions the presence of T4 NrdD with NrdG revealed a strong doublet EPR signal (g = 2.0039). Isotope labeling of the NrdD with [2H]glycine and [13C]glycine, respectively, confirmed the presence of a stabilized glycine radical. The unpaired electron is strongly coupled to C-2 in glycine and the doublet splitting originates from one of the alpha-protons. The glycine residue at position 580 was determined to be the radical containing residue through site-directed mutagenesis studies involving a G580A NrdD mutant. The glycyl radical generation was specific for the T4 NrdG, and the host E. coli NrdG was found to be unable to activate the phage reductase. Finally, anaerobic purification revealed the holoenzyme complex to contain iron, whereas the NrdD polypeptide was found to lack the metal. Our results suggest a tetrameric structure for the T4 anaerobic ribonucleotide reductase containing one homodimer each of NrdD and NrdG, with a single glycyl radical present.
Young
P
P
Department of Molecular Biology, Stockholm University, S-10691 Stockholm, Sweden.
Andersson
J
J
Sahlin
M
M
Sjöberg
B M
BM
D000693:Anaerobiosis; D001426:Bacterial Proteins; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins; D005609:Free Radicals; D005998:Glycine / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology*
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D005609:Free Radicals; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014764:Viral Proteins; C490362:NrdG protein, E coli; D012264:Ribonucleotide Reductases; C101141:anaerobic ribonucleotide reductase; C490361:nrdG protein, Enterobacteria phage T4; C090389:nrdG protein, bacteria; D005998:Glycine