Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Abstract

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) . These results present a structural and biochemical characterization of the active RNR complex "trapped" during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.conformational equilibria | radical transfer | unnatural amino acid R ibonucleotide reductase (RNR) is the sole enzyme responsible for the conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleoside diphosphates (dNDPs), providing the cell with the monomeric precursors necessary for DNA replication and repair (1, 2). The class I RNRs are composed of two subunits, α and β;the active form of the prototypical Escherichia coli class Ia enzyme is generally accepted to be α2β2 (3, 4). The α2 subunit houses the active site, where the four NDP substrates (CDP, ADP, GDP, and UDP) are reduced, and two distinct regulatory sites, where allosteric effectors (ATP, dGTP, dTTP, and dATP) bind. The specificity site dictates which of the four substrates is reduced, whereas the activity site binds ATP/dATP and regulates the overall rate of reduction (5). The β2 subunit, an obligate dimer, contains a diferric-tyrosyl radical cofactor (Y 122 •) that is essential for catalysis. Xray crystal structures of the individual E. coli β2 and α2 subunits have been solved (6, 7). However, the weak interaction between α2 and β2 (8), the conformational rearrangements induced by nucleotide binding (2, 9, 10), and complicated subunit equilibria (11) have precluded detailed structural characterization of any active RNR complexes. We now report the characterization of an active, kinetically stable α2β2 complex that forms transiently during turnover.Nearly 2 decades ago, Uhlin and Eklund (7) put forth a docking model for the active E. coli α2β2 complex based on shape complementarity between the structures of the individual subunits.Their model predicted a 35-Å distance between the diferric-Y 122 • cofactor in β2 and the active site cysteine (C 439 ) in α2, the transient oxidation of which is a prerequisite for nucleotide reduction (1). A radical transfer (RT) pathway of conserved aromatic amino acids was proposed to account for kinetically competent radical propagation over this long distance (7). The thermodynamics of Y oxidation require loss of a proton to accompany loss of an electron, and the more detailed mechanism for proton-coupled electron transfer shown in Fig. 1A has emerged from experiments conducted in our laboratories (12,13).Evidence for the utilization of an amino acid pathway in longrange RT has been derived from several types of experiments. Initial site-directed mutagenesis studies of the conserved residues (Fig. 1A) supported t...

Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase

“…Size exclusion chromatography (SEC) was performed in tandem with SAXS data collection in order to eliminate any aggregated protein from each sample (Figure S3). Scattering data was collected at 5 s intervals from the column eluate and inspected for heterogeneity using Kratky analysis, a method commonly employed for detecting conformational changes due to ligand binding events (44; 45), multimerization (46-48), and flexibility (49). Every five consecutive datasets were averaged and transformed into Kratky plots.…”

A Fluorimetric Readout Reporting the Kinetics of Nucleotide‐Induced Human Ribonucleotide Reductase Oligomerization

“…The α 2 dimeric state is induced upon effector binding at the S site [6,8–14] and dimerization is a prerequisite for the adoption of the α 2 β 2 active state by the holoenzyme. [25] The α 2 quaternary state of hRNR-α (without β) has also been well-characterized by crystallography. [10] We were thus interested in the extent to which our fluorescence protocol could distinguish between α hexamerization and dimerization.…”

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase

“…8–10 The α 2 subunit contains the catalytic site, the thiyl radical forming cysteine residue (C 439 ), a nucleoside diphosphate (NDP) substrate binding site, a specificity site that binds (deoxy)nucleosides triphosphates (NTP/dNTP) and controls which NDP is reduced, and an activity site that binds adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP), whose ratio governs the overall rate of the reaction. 9 The β 2 subunit contains a stable diiron-tyrosyl radical cofactor (Fe 2 III -O/Y 122 •) essential for C 439 • generation in α 2 >35 Å away.…”