Bacteriophage T4 Anaerobic Ribonucleotide Reductase Contains a Stable Glycyl Radical at Position 580

Abstract

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 [ H]glycine and [13 C]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 ␣-protons. The glycine residue at position 580 was determined to be the radical containing residue through sitedirected 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.

Cysteines Involved in Radical Generation and Catalysis of Class III Anaerobic Ribonucleotide Reductase

“…2). The full-length NrdD protein migrates approximately according to its theoretical molecular mass of 68 kDa (21). The truncated form, denoted NrdDЈ, migrates slightly faster than the full-length NrdD, in good agreement with the anticipated truncation at Gly-580 ϳ3 kDa from the C terminus of the phage T4 NrdD.…”

“…Plasmids-Plasmids pET29T4nrdD and pET21aT4nrdG were described previously (21). Plasmid pEE1010 containing the gene for ferredoxin (flavodoxin) NADP ϩ reductase (22) was a kind gift from Vera Bianchi and Elisabeth Haggård-Ljungquist.…”

“…Oligonucleotide-directed Mutagenesis-All mutants were constructed with the Kunkel method (24, 25) using the Mutagene phagemid in vitro mutagenesis kit (version 2) as described previously (21). The mutagenic oligonucleotides also contained the insertion or deletion of a restriction enzyme cleavage site to facilitate the screening for mutants.…”

“…21 was followed. Anaerobic activity assays and EPR measurements in anaerobic crude extracts are also described in Ref.…”

“…We have earlier used site-directed mutagenesis to identify Gly-580 in phage T4 class III RNR as the site of the stable glycyl radical (21). In this work we have used the same approach to identify cysteine residues involved in the reaction mechanism of the T4 class III enzyme.…”