Structural investigation of 2'-azido-2'-deoxyribonucleoside-5'-diphosphate inhibition of class Ia Escherichia coli ribonucleotide reductase.

Abstract

Bacterial class Ia ribonucleotide reductases (RNRs) are attractive antibiotic targets due to their essential role in the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. The catalytically active state of class Ia RNRs consists of a heterodimer of two homodimers, α;2 and β;2, in which the α;2 subunit contains substrate-binding and allosteric sites and the β;2 subunit contains two di-iron sites responsible for the initial steps of a 32-Å radical transfer pathway that enables conversion of the ribonucleotide substrate. Recent structural work in the Drennan lab has elucidated the active state structure of this enzyme, but required a doubly-mutated variant of β;2, E52Q/F3Y122-β;2, to decrease the rate of subunit dissociation. We describe here the use of 2′-azido-2′-deoxyribonucleoside-5′-diphosphate, a nucleotide analog, to trap the active state structure of the wild type Escherichia coli class Ia RNR and subsequent structural efforts to elucidate the conformational trajectory of the dissociation. This work provides a basis for understanding the complex regulatory mechanisms of RNR, which may result in novel antibiotic targets.