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Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli class Ia ribonucleotide reductase.

ABSTRACT: Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an ?2 subunit that houses the active site and allosteric regulatory sites and a ?2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact ?2?2 state allowing for radical transfer from ?2 to ?2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on ? promotes formation of an ?4?4 ring-like state. Here, we investigate whether the ?4?4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the ?-? interface (S37D/S39A-?2, S39R-?2, S39F-?2, E42K-?2, or L43Q-?2) that disrupt the ?4?4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that ?4?4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the ?-? interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the ?-? interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-?2 or E42K-?2) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.

SUBMITTER: Chen PY

PROVIDER: S-EPMC6028975 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli class Ia ribonucleotide reductase.

Chen Percival Yang-Ting PY Funk Michael A MA Brignole Edward J EJ Drennan Catherine L CL

The Journal of biological chemistry 20180426 26

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α2 subunit that houses the active site and allosteric regulatory sites and a β2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2β2 state allowing for radical transfer from β2</sub ...[more]

PMID: 29700111

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Project description:Ribonucleotide reductases (RNRs) catalyze the conversionof nucleotides to 2'-deoxynucleotides and are classified on the basis of the metallo-cofactor used to conduct this chemistry. The class Ia RNRs initiate nucleotide reduction when a stable diferric-tyrosyl radical (Y•, t1/2 of 4 days at 4 °C) cofactor in the ?2 subunit transiently oxidizes a cysteine to a thiyl radical (S•) in the active site of the ?2 subunit. In the active ?2?2 complex of the class Ia RNR from E. coli , researchers have proposed that radical hopping occurs reversibly over 35 Å along a specific pathway comprised of redox-active aromatic amino acids: Y122• ? [W48?] ? Y356 in ?2 to Y731 ? Y730 ? C439 in ?2. Each step necessitates a proton-coupled electron transfer (PCET). Protein conformational changes constitute the rate-limiting step in the overall catalytic scheme and kinetically mask the detailed chemistry of the PCET steps. Technology has evolved to allow the site-selective replacement of the four pathway tyrosines with unnatural tyrosine analogues. Rapid kinetic techniques combined with multifrequency electron paramagnetic resonance, pulsed electron-electron double resonance, and electron nuclear double resonance spectroscopies have facilitated the analysis of stable and transient radical intermediates in these mutants. These studies are beginning to reveal the mechanistic underpinnings of the radical transfer (RT) process. This Account summarizes recent mechanistic studies on mutant E. coli RNRs containing the following tyrosine analogues: 3,4-dihydroxyphenylalanine (DOPA) or 3-aminotyrosine (NH2Y), both thermodynamic radical traps; 3-nitrotyrosine (NO2Y), a thermodynamic barrier and probe of local environmental perturbations to the phenolic pKa; and fluorotyrosines (FnYs, n = 2 or 3), dual reporters on local pKas and reduction potentials. These studies have established the existence of a specific pathway spanning 35 Å within a globular ?2?2 complex that involves one stable (position 122) and three transient (positions 356, 730, and 731) Y•s. Our results also support that RT occurs by an orthogonal PCET mechanism within ?2, with Y122• reduction accompanied by proton transfer from an Fe1-bound water in the diferric cluster and Y356 oxidation coupled to an off-pathway proton transfer likely involving E350. In ?2, RT likely occurs by a co-linear PCET mechanism, based on studies of light-initiated radical propagation from photopeptides that mimic the ?2 subunit to the intact ?2 subunit and on [(2)H]-ENDOR spectroscopic analysis of the hydrogen-bonding environment surrounding a stabilized NH2Y• formed at position 730. Additionally, studies on the thermodynamics of the RT pathway reveal that the relative reduction potentials decrease according to Y122 < Y356 < Y731 ? Y730 ? C439, and that the pathway in the forward direction is thermodynamically unfavorable. C439 oxidation is likely driven by rapid, irreversible loss of water during the nucleotide reduction process. Kinetic studies of radical intermediates reveal that RT is gated by conformational changes that occur on the order of >100 s(-1) in addition to the changes that are rate-limiting in the wild-type enzyme (?10 s(-1)). The rate constant of one of the PCET steps is ?10(5) s(-1), as measured in photoinitiated experiments.

Dataset Information

Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli class Ia ribonucleotide reductase.

Publications

Disruption of an oligomeric interface prevents allosteric inhibition of <i>Escherichia coli</i> class Ia ribonucleotide reductase.

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