Bacteriophage T4 gene 55.9 encodes an activity required for anaerobic ribonucleotide reduction

Bacteriophage T4 contains a phage-encoded anaerobic ribonucleoside triphosphate reductase, nrdD, previously named sunY. An open reading frame, 55.9, that resides downstream of the phage reductase was observed to have amino acid sequence similarity with the E. coli pyruvate formate-lyase (Pfl) activating enzyme. A stop codon was engineered into the cloned 55.9 gene and then recombined back into the phage genome. Phage-infected extracts that lack a functional 55.9 product have a 6-fold reduction in anaerobic ribonucleotide reductase activity and are unable to activate overexpressed T4 NrdD. Restoration of reductase activity was possible when 55.9- and nrdD- T4-infected Escherichia coli extracts were conjointly assayed. Comparing the anaerobic burst size of 55.9- infections to that of the parental phage indicates that anaerobic de novo synthesis of deoxyribonucleotides is nearly abolished in phage lacking the 55.9 product. We propose that T4 55.9 encodes an enzyme that activates T4 NrdD by generating a glycyl radical in the phage-encoded reductase. The homology between the Pfl activating enzyme and T4 55.9 product (in this communication renamed NrdG) in function as well as amino acid sequence is presumably a remnant of an ancient heritage between Pfl and the anaerobic ribonucleotide reductases.