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Riboswitches01:56

Riboswitches

Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases
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Structural basis for activation of class Ib ribonucleotide reductase.

Amie K Boal1, Joseph A Cotruvo, JoAnne Stubbe

  • 1Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

Science (New York, N.Y.)
|August 7, 2010
PubMed
Summary

Class Ib ribonucleotide reductase in E. coli uses either manganese or iron cofactors. Structural studies reveal distinct activation pathways and a key channel for manganese cofactor assembly, involving the NrdI protein.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Class Ib ribonucleotide reductase (RNR) from Escherichia coli utilizes a Mn(III)2-tyrosyl radical (Y•) or Fe(III)2-Y• cofactor for nucleotide reduction.
  • Iron cofactor assembly is spontaneous with O2, but manganese cofactor activation requires the reduced flavoprotein NrdI.

Purpose of the Study:

  • To elucidate the structural mechanisms underlying cofactor activation in class Ib RNR.
  • To investigate the role of NrdI in the assembly of the manganese cofactor.

Main Methods:

  • X-ray crystallography of E. coli Mn(II)2-NrdF and Fe(II)2-NrdF.
  • Structural analysis of Mn(II)2-NrdF in complex with reduced and oxidized NrdI.
  • Crystallographic detection of reaction intermediates.

Main Results:

  • Distinct coordination environments were observed for Mn(II)2-NrdF and Fe(II)2-NrdF, suggesting different oxidant binding sites.
  • A continuous channel was identified connecting the NrdI flavin cofactor to the Mn(II)2 active site in Mn(II)2-NrdF.
  • A putative peroxide intermediate was detected within this channel, supporting the proposed catalytic mechanism.

Conclusions:

  • The findings reveal distinct structural pathways for the activation of manganese and iron cofactors in class Ib RNR.
  • The NrdI-mediated manganese cofactor assembly involves a direct channel facilitating oxidant transfer and intermediate formation.