Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Abstract

Escherichia coli obtains the deoxyribonucleotides required for DNA synthesis from ribonucleotides through the activity of the enzyme ribonucleoside diphosphate reductase (1-3). A closely related enzyme has the same function in mammalian cells. Both enzymes consist of two nonidentical subunits, proteins B1 and B2 (4) of E. coli and the corresponding mammalian proteins Ml and M2 (5). B2 and M2 contain a tyrosyl radical (6-8) that has arisen by oxidation of a specific tyrosine residue of the polypeptide chain. An oxygenrequiring enzyme system catalyzes the formation of this radical in E. coli (9, 10). However, E. coli grows well also under anaerobic conditions. This poses the question how deoxyribonucleotides are synthesized in the absence of oxygen. One could imagine that during anaerobiosis an electron acceptor other than oxygen might function in radical generation. Or, deoxyribonucleotides might be formed by other enzymes. The second alternative was favored strongly from genetic experiments (11-13) demonstrating that mutations in the nrdA or nrdB gene, coding for protein B1 or B2, did not affect the anaerobic growth of the bacteria. These experiments did, however, not distinguish between a different ribonucleotide reductase or a completely different mode of synthesis-e.g., via the deoxyribose 5-phosphate aldolase pathway (15).Here we first establish from isotope experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. We then describe the existence of a different reductase in extracts from anaerobically grown bacteria. The enzyme is not inhibited by antibodies raised against protein B1 or B2 and is only moderately sensitive to hydroxyurea, a drug that strongly inhibits the aerobic reductase, but is rapidly inactivated by air. The substrate of the reaction appears to be CTP and not CDP, as for the aerobic reaction. MATERIALS AND METHODSBacterial Strains. The E. coli K-12 strain AB1157 and its oxygen-sensitive derivative Oxys-11 (11) were obtained from H. I. Adler (Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN). E. coli So1452, a strain with an insertion mutation in the gene for cytidine deaminase, was a gift from B. Mygind (Institute for Biological Chemistry, University of Copenhagen, Denmark).Preparation of Bacterial Extracts. Five-liter cultures of AB1157 or Oxys-11 were grown anaerobically in Luria broth containing 1 g of glucose per liter under continuous bubbling with 4% C02/96% N2 in a Microferm New Brunswick fermentor with constant pH control. When the bacteria had reached midlogarithmic phase (OD = 1.0 at 640 nm) they were harvested by centrifugation in twelve 0.5-liter bottles in cold Sorvall centrifuges, resuspended under a flow of argon in a small volume of an anaerobic 50 mM Tris buffer (pH 7.5), and transferred under argon to four preweighed capped Beckman no. 355603 thick-walled polycarbonate tubes. After centrifugation and removal of the supernatant solution, the weighed bacteria (1-2 g per tube) were stored under liquid...

Ribonucleotide Reductases

“…Class II and class III reductases have not been found in the same organism. In eubacteria, class III exists together with class I in facultative anaerobes (10,13,18). Among strict anaerobes only methanobacteria have been found to contain a class III enzyme (15).…”

“…Depending on the allosteric configuration, one of the four common ribonucleotides binds to the catalytic site (34,36). Ribonucleoside diphosphates are the substrates for class I enzymes, whereas the two class III enzymes investigated so far use triphosphates (10,14). The prototype class II L. leishmannii enzyme also uses triphosphates (38), but other, later discovered members of this class use diphosphates (24,(39)(40)(41).…”

“…With one (51), or possibly two (52), exceptions they have not been found in eukaryotes. Class III enzymes are used by facultative anaerobic eubacteria during anaerobic growth (10,13,14) and by anaerobic archaebacteria (15).…”

“…The discovery of a second ribonucleotide reductase in anaerobically growing E. coli (10) came as no surprise, as the generation of the tyrosyl radical of class I enzymes requires oxygen for its formation. The anaerobic enzyme is also a protein radical, with the unpaired electron located on a glycyl residue at the COOH-terminal region of the polypeptide chain (11).…”

Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein

“…During anaerobic growth, Escherichia coli induces an enzyme that catalyzes the reduction of CTP to dCTP (7)(8)(9)11). The gene for this enzyme was recently cloned (28) and found to be distinct from nrdA and nrdB, which code for the aerobic ribonucleoside diphosphate reductase (29).…”