[20] Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase

Jm, Bollinger; Wh, Tong; Ravi, Natarajan; Bh, Huynh; De, Edmondson; Ja, Stubbe

doi:10.1016/0076-6879(95)58052-2

Cited by 83 publications

(105 citation statements)

References 15 publications

Supporting

Mentioning

103

Contrasting

Order By: Relevance

“…Because most known di-iron enzymes are catalytically active in a reduced (diferrous) form (12, 13), we first used chemical reductants to prepare diferrous AurF for singleturnover experiments. Based on the protocols of similar experiments developed for the hydroxylase subunit of the soluble methane monooxygenase (MMOH) (14,15), we used either sodium dithionite or ascorbate as a chemical reductant and various chemicals, including phenazine methosulfate (PMS), methyl viologen, and proflavin as an electron mediator. It was found that the in vitro enzymatic activity of AurF was best reconstituted in the presence of PMS and ascorbate [supporting information (SI) Fig.…”

Section: Resultsmentioning

confidence: 99%

In vitro reconstitution and crystal structure of p -aminobenzoate N -oxygenase (AurF) involved in aureothin biosynthesis

Choi¹,

Zhang

Brunzelle

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

143

216

View full text Add to dashboard Cite

p-aminobenzoate N-oxygenase (AurF) from Streptomyces thioluteus catalyzes the formation of unusual polyketide synthase starter unit p-nitrobenzoic acid (pNBA) from p-aminobenzoic acid (pABA) in the biosynthesis of antibiotic aureothin. AurF is a metalloenzyme, but its native enzymatic activity has not been demonstrated in vitro, and its catalytic mechanism is unclear. In addition, the nature of the cofactor remains a controversy. Here, we report the in vitro reconstitution of the AurF enzyme activity, the crystal structure of AurF in the oxidized state, and the cocrystal structure of AurF with its product pNBA. Our combined biochemical and structural analysis unequivocally indicates that AurF is a non-heme di-iron monooxygenase that catalyzes sequential oxidation of aminoarenes to nitroarenes via hydroxylamine and nitroso intermediates.di-iron enzymes ͉ metalloenzymes ͉ N-oxygenation ͉ reaction mechanism

show abstract

Section: Resultsmentioning

confidence: 99%

In vitro reconstitution and crystal structure of p -aminobenzoate N -oxygenase (AurF) involved in aureothin biosynthesis

Choi¹,

Zhang

Brunzelle

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

143

216

View full text Add to dashboard Cite

show abstract

“…Acquisition parameters were as described previously (19,43,48) at 9.4 GHz, 2 milliwatt power, 2.52 ϫ 10 3 gain, 1.5 G modulation amplitude, and 100 kHz modulation frequency. Spin quantitation was performed by double integration of the signal and comparison with an E. coli NrdB sample in which the Tyr ⅐ content had been determined by the drop line method using UV-visible spectroscopy (49). Analysis was carried out using WinEPR software (Brüker).…”

Section: Whole-cell Epr Spectroscopy Of Galrnr4 Cells and Epr Spectromentioning

confidence: 99%

Investigation of in Vivo Diferric Tyrosyl Radical Formation in Saccharomyces cerevisiae Rnr2 Protein

Zhang

Liu

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Yeast RNR small subunit is an Rnr2-Rnr4 heterodimer; only Rnr2 contains a cluster. Results: rnr4 and dre2 mutants are defective in Rnr2 cluster formation and display synthetic growth defects with grx3/4. Conclusion: Rnr4 stabilizes Rnr2 for cluster assembly via a pathway dependent on monothiol glutaredoxins Grx3/Grx4 and Fe-S cluster protein Dre2. Significance: Understanding RNR cluster assembly may provide new cancer therapeutic strategy.

show abstract

“…The Y• of E. coli R2 (2.5-115 µM) was used to generate a standard curve. The concentration of Y• in the E. coli standard was determined by the drop-line correction method (19), and the protein concentration was determined using the known ε 280 of 131 mM −1 cm −1 . For determination of the Y• concentration in vivo, the EPR spectrum of yeast cells treated with hydroxyurea (150 mM, 1 h) was subtracted from that of untreated cells to subtract contributions from species other than the Y• of RNR as described (20).…”

Section: In Vivo Concentration Of Y• By Whole-cell Epr Spectroscopymentioning

confidence: 99%

The Active Form of the Saccharomyces cerevisiae Ribonucleotide Reductase Small Subunit Is a Heterodimer in Vitro and in Vivo

Perlstein

Ge²,

Ortigosa³

et al. 2005

Biochemistry

Self Cite

View full text Add to dashboard Cite

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y•) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (β 2 ) and Y4 (β′ 2 ). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (ββ′) is thought to be the active form, several rnr4Δ strains are viable. To resolve this paradox, N-terminally epitope-tagged β and β′ were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His 6 )-β 2 ( His β 2 ) is mixed with β′ 2 , apo-His ββ′ forms quantitatively within 2 min. In contrast, holo-ββ′ fails to exchange with apo-His β 2 to form holo-His ββ and β′ 2 . Isolation of genomically encoded tagged β or β′ from yeast extracts gave a 1:1 complex of β and β′, suggesting that ββ′ is the active form. The catalytic activity, protein concentrations, and Y• content of the rnr4Δ and wild type (wt) strains were compared to clarify the role of β′ in vivo. The Y• content of rnr4Δ is 15-fold less than that of wt, consistent with the observed low activity of rnr4Δ extracts (<0.01 nmol min −1 mg −1 ) versus wt (0.06 ± 0.01 nmol min −1 mg −1 ). FLAG β 2 isolated from the rnr4Δ strain has a specific activity of 2 nmol min −1 mg −1 , similar to that of reconstituted apo-His β 2 (10 nmol min −1 mg −1 ), but significantly less than holo-His ββ′ (~2000 nmol min −1 mg −1 ). These studies together demonstrate that β′ plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is ββ′.Ribonucleotide reductases (RNRs) 1 catalyze the conversion of ribonucleotides to deoxyribonucleotides, providing the monomeric precursors for DNA replication and repair (1). The class I RNRs are composed of a large subunit, R1, and a small subunit, R2. R1 contains the site of nucleotide reduction and the allosteric effector binding sites that control the rate and the specificity of nucleotide reduction. R2 houses the diferric-tyrosyl radical † D.L.P. and A.D.O. were supported in part by the NIH training grant 5T32 CA 09112-28. J.S. acknowledges support of the NIH (GM29595). M.H. acknowledges support of the NIH (CA095207) and the ACS (0305001GMC).

show abstract

[20] Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase

Cited by 83 publications

References 15 publications

In vitro reconstitution and crystal structure of p -aminobenzoate N -oxygenase (AurF) involved in aureothin biosynthesis

In vitro reconstitution and crystal structure of p -aminobenzoate N -oxygenase (AurF) involved in aureothin biosynthesis

Investigation of in Vivo Diferric Tyrosyl Radical Formation in Saccharomyces cerevisiae Rnr2 Protein

The Active Form of the Saccharomyces cerevisiae Ribonucleotide Reductase Small Subunit Is a Heterodimer in Vitro and in Vivo

Contact Info

Product

Resources

About