Escherichia coli ribonucleotide reductase. Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme.

Karlsson, Matts; Sahlin, Margareta; Sjoberg, B.M.

doi:10.1016/s0021-9258(18)42322-8

Cited by 60 publications

(43 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Y122· decay is visualized as a flattening of a peak at 410 nm in the UV–vis spectrum of β 2 (Figure A,B) without affecting features at 325 and 370 nm, which are attributed to the differic cluster. Experiments using 20 μM 3AP-Fe(II) complex quenched Y122· with a rate constant ( k ) of 32.6 M –1 s –1 , at least 60 times faster than the inhibition of E. coli β 2 by HU, which we measured as k = 0.42 M –1 s –1 (Figure C, Table ), similar to earlier reports. ,, The broad feature at ∼350–430 nm in the presence of 3AP-Fe(II) (Figure B), compared to HU (Figure A), is consistent with overlapping contributions of the β 2 cluster and the 3AP-Fe(II) inhibitor, based on model spectra (Figure S2, purple trace). Neither 3AP nor 3AP-Fe(III) quenched Y122· (Figure C, Figure S1A–D).…”

supporting

confidence: 91%

“…Class Ia RNRs are inactivated by radical scavengers. For example, hydroxyurea (HU; Scheme ) quenches Y· in isolated β 2 (Scheme , Figure ) and scavenges tyrosyl radicals along the PCET pathway during catalysis. , Despite its continued clinical use as a broad-spectrum chemotherapeutic, HU is associated with significant side effects likely because HU is nonspecific and therefore must be used at high concentrations . Studies have also suggested that RNR may not be the functional target for cytotoxicity of HU in vivo . − Other clinically relevant radical scavenger inhibitors include 4-methoxyphenol (4MP).…”

mentioning

confidence: 99%

See 1 more Smart Citation

E. coli Ribonucleotide Reductase β2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine–Fe(II) Adduct

Safiarian

Watson

Lieberman

et al. 2021

J. Phys. Chem. Lett.

View full text Add to dashboard Cite

Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR β 2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122• of E. coli RNR β 2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the β 2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity (K d ∼ 6 nM) of 3AP-Fe(II) for β 2 . Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.

show abstract

supporting

confidence: 91%

mentioning

confidence: 99%

E. coli Ribonucleotide Reductase β2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine–Fe(II) Adduct

Safiarian

Watson

Lieberman

et al. 2021

J. Phys. Chem. Lett.

View full text Add to dashboard Cite

show abstract

“…the Tyr-122 resides within a hydrophobic pocket 10 Å from the surface and is apparently inaccessible to solvent. The regulatory state of the enzyme affects radical stability as well, with a large decrease in stability of the active enzyme complex as opposed to R2 alone (31). These data provide support for the binding of HU to residues other than Tyr-122; indeed, multiple binding sites for HU may exist on both the R2 and R1 proteins.…”

Section: Discussionmentioning

confidence: 66%

“…In the absence of HU or other scavengers, the R2 radical has an extremely long half-life, surviving for days at room temperature or for months stored at Ϫ80°C. Scavengers like HU can efficiently abstract the radical, however, in a manner dependent on the regulatory state of the enzyme (31). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Mutations in the R2 Subunit of Ribonucleotide Reductase That Confer Resistance to Hydroxyurea

Sneeden

Loeb

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.

show abstract

“…The larger the value of the *E, the weaker is the acid. The gas-phase basicity was deÐned as the energy of protonation (*E) for reaction (4).…”

Section: Computational Detailsmentioning

confidence: 99%

Molecular structure and gas-phase reactivity of zileuton and its N-dehydroxylated metabolite: two-layered ONIOM calculations

Remko¹,

Lyne²,

Richards³

2000

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

Escherichia coli ribonucleotide reductase. Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme.

Cited by 60 publications

References 34 publications

E. coli Ribonucleotide Reductase β2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine–Fe(II) Adduct

E. coli Ribonucleotide Reductase β2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine–Fe(II) Adduct

Mutations in the R2 Subunit of Ribonucleotide Reductase That Confer Resistance to Hydroxyurea

Molecular structure and gas-phase reactivity of zileuton and its N-dehydroxylated metabolite: two-layered ONIOM calculations

Contact Info

Product

Resources

About