Dataset Information

Glutamate 52-? at the ?/? subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

ABSTRACT: Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of ? and ? subunits that form a transient, active ?2?2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (?/?) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of ?2?2 created from X-ray structures of ? and ? and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the ?/? interface (Glu350 and Glu52 in ?2 and Arg329 and Arg639 in ?2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-?2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F3Y122•) replacing the stable Tyr122• in WT-?2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His6-?2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr356•-?2), 0.5 eq of dCDP/F3Y122•, and formation of an ?2?2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the ?2?2 complex formed.

SUBMITTER: Lin Q

PROVIDER: S-EPMC5454104 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Glutamate 52-β at the α/β subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Lin Qinghui Q Parker Mackenzie J MJ Taguchi Alexander T AT Ravichandran Kanchana K Kim Albert A Kang Gyunghoon G Shao Jimin J Drennan Catherine L CL Stubbe JoAnne J

The Journal of biological chemistry 20170404 22

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway ...[more]

PMID: 28377505

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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

Glutamate 52-? at the ?/? subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Publications

Glutamate 52-β at the α/β subunit interface of <i>Escherichia coli</i> class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Similar Datasets

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