Importance of the maintenance pathway in the regulation of the activity of Escherichia coli ribonucleotide reductase

Daniela Hristova, Chia Hung Wu, Wei Jiang, Carsten Krebs, JoAnne Stubbe

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α1β 2 active complex. β contains the diferric tyrosyl radical (Y) cofactor that is essential for the reduction process that occurs on a. [Y] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an L-arabinose promoter were constructed. Using these constructs and with [L-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 μM. [Y] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1-0.3 Y radical per β. To determine if the substoichiometric Y level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O2 to assemble the cofactor and EPR analysis to quantitate Y, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 μM. These titrations, furthermore, resulted in 1 Y radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y in vivo in E. coli is a mechanism of regulating RNR activity.

Original languageEnglish (US)
Pages (from-to)3989-3999
Number of pages11
JournalBiochemistry
Volume47
Issue number13
DOIs
StatePublished - Apr 1 2008

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Ribonucleotide Reductases
Escherichia coli
Maintenance
Arabinose
Titration
Paramagnetic resonance
Ferredoxins
Growth
Cell Extracts
Complex Mixtures
Spectrum Analysis
Nucleotides
Iron
Modulation
Spectroscopy

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Hristova, Daniela ; Wu, Chia Hung ; Jiang, Wei ; Krebs, Carsten ; Stubbe, JoAnne. / Importance of the maintenance pathway in the regulation of the activity of Escherichia coli ribonucleotide reductase. In: Biochemistry. 2008 ; Vol. 47, No. 13. pp. 3989-3999.
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abstract = "Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α1β 2 active complex. β contains the diferric tyrosyl radical (Y•) cofactor that is essential for the reduction process that occurs on a. [Y•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an L-arabinose promoter were constructed. Using these constructs and with [L-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 μM. [Y•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1-0.3 Y• radical per β. To determine if the substoichiometric Y• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O2 to assemble the cofactor and EPR analysis to quantitate Y•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 μM. These titrations, furthermore, resulted in 1 Y• radical per β, the highest levels reported. Whole cell M{\"o}ssbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y• in vivo in E. coli is a mechanism of regulating RNR activity.",
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Importance of the maintenance pathway in the regulation of the activity of Escherichia coli ribonucleotide reductase. / Hristova, Daniela; Wu, Chia Hung; Jiang, Wei; Krebs, Carsten; Stubbe, JoAnne.

In: Biochemistry, Vol. 47, No. 13, 01.04.2008, p. 3989-3999.

Research output: Contribution to journalArticle

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AB - Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α1β 2 active complex. β contains the diferric tyrosyl radical (Y•) cofactor that is essential for the reduction process that occurs on a. [Y•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an L-arabinose promoter were constructed. Using these constructs and with [L-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 μM. [Y•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1-0.3 Y• radical per β. To determine if the substoichiometric Y• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O2 to assemble the cofactor and EPR analysis to quantitate Y•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 μM. These titrations, furthermore, resulted in 1 Y• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y• in vivo in E. coli is a mechanism of regulating RNR activity.

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