A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.

Abstract

The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the Rl and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S.typhimurium nrdE and nrdF genes (RlE and R2F Ribonucleotide reductases catalyze the synthesis of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis. At least three separate classes of enzymes are known, each with a distinct protein structure but all requiring a protein radical for catalysis (1). The long-studied aerobic Escherichia coli enzyme is the prototype for class I enzymes, also present in all higher organisms and some other microorganisms. E. coli genes nrdA and nrdB encode the a and 8 polypeptide chains, respectively, that form the Rl (a2) and R2 (132) proteins that constitute the enzyme (2). Each protomer of the Rl dimer (2 x 85.7 kDa) contains one substrate-binding site with redox-active thiols involved in the reduction of the substrate ribonucleoside diphosphate, and two separate types of allosteric sites: one, the activity site, controls the overall activity of the enzyme, with ATP as a positive effector and dATP as a negative effector; the other, the substrate-specificity site, controls the specificity of the enzyme, with ATP and dATP favoring pyrimidine reduction, dTTP favoring GDP reduction, and dGTP favoring ADP reduction (3).The R2 dimer (2 x 43.4 kDa) contains two dinuclear iron centers with associated stable tyrosyl free radicals, located at Tyr-122 of the polypeptide chain (4). The drug hydroxyurea scavenges this radical and thereby inactivates the enzyme. Class II and III enzymes lack the tyrosyl radical. Class II enzymes, with the Lactobacillus leichmanni enzyme as a prototype, employ adenosylcobalamin as a radical generator, whereas class III enzymes use S-adenosylmethionine together with iron for this purpose.Salmonella typhimurium contains an active class I enzyme with amino acid sequences 96.5% and 98.4% identical to the E. coli Rl and R2 proteins, respectively (A.J., unpublished results). Recent genetic evidence involving complementation of nrd mutants of E. coli suggested the presence in S. typhimurium of the genes, nrdE and nrdF, coding for a second class I enzyme (5). These genes are also present on the chromosome of E. coli but are, under standard growth conditions, expressed only when present on a plasmid. The amino acid sequences deduced for the corresponding proteins showed a limited identity with other class I enzymes but contained many of their catalytically important residues.We have purified and characterized the two proteins encoded by the cloned genes nrdE and nrdF from S. typhimurium. Each protein is a homodimer. Together they catalyze the reductio...

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

“…Our recent studies of B. subtilis class Ib RNR showed that dATP inhibits RNR activity at concentrations Ͼ5 M (47). This result was surprising because class Ib RNRs do not have an ATP cone or activity domain, the typical binding site for dATP that facilitates class Ia RNR inhibition (52,60,61). To determine whether the S. sanguinis class Ib RNR is inhibited by dATP, the activity of Mn Y ⅐ -NrdF was measured in the presence of increasing dATP concentrations (Fig.…”

Alternative oxygen‐dependent and oxygen‐independent ribonucleotide reductases in Streptomyces: cross‐regulation and physiological role in response to oxygen limitation

“…Repeated trials failed to generate any authentic mutants implying that deletion of both genes is lethal and that S. coelicolor does not possess any other functional RNR systems (indeed, no other genes encoding RNR-like proteins are present in the complete genome sequences of S. coelicolor and Streptomyces avermitilis ). Correlation of this conclusion was obtained from a study of the effect of hydroxyurea -a potent inhibitor of class I RNRs (Sinha and Snustad, 1972;Jordan et al ., 1994) -on the growth of M145 and the nrdB and nrdJ deletion mutant strains. Figure 2 shows that all three strains, M145, M145 D nrdB::apr and M145 D nrdJ::apr , grew equally well on solid medium whereas the M145 D nrdJ::apr strain alone failed to grow in the presence of 10 mM hydroxyurea.…”

NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

“…coli NrdE and NrdF have not been previously purified, although the equivalent proteins from Salmonella typhimurium, sharing 89 and 87% sequence identity, respectively, have been purified (10) and their structures have been determined (11,12). Efforts to purify NrdI have been reported but were hampered by poor solubility.…”