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Florence Luttringer

The anaerobic ribonucleotide reductase from Escherichia coli, a zinc protein. Importance of the metal-binding site for the enzyme structure and activation

Published on 10 November 2006


Thesis presented November 10, 2006

Abstract:
Ribonucleotide reductases (RNRs) are critical enzymes for the de novo synthesis of DNA in all organisms. For its anaerobic growth, Escherichia coli depends on a class III RNR, which harbors in its active form an oxygen-sensitive free radical located on the Gly681 residue, the formation of which involves the concerted action of four components: an act​ivating iron-sulfur protein, S-adenosylmethionine, dithiothreitol (DTT), and a reducing system. A Zn(Cys)4 center has recently been found in the C-terminal region of the crystal structure of the RNR from bacteriophage T4. In this work we define new conditions allowing for the preparation of a high specific activity reductase. We show that the zinc site controls the structuration of the C-ter loop carrying the radical site. Finally we demonstrate that DTT is not essential for radical formation but rather acts on radical transfer reactions between Gly and the substrate.

Keywords:
Enzymatic catalysis, radical, zinc, proteolysis

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