Three-dimensional structure of the free radical protein of ribonucleotide reductase

Nordlund, P.; Sjöberg, Britt‐Marie; Eklund, Hans

doi:10.1038/345593a0

Cited by 851 publications

(741 citation statements)

References 54 publications

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“…This fold is also found in the R2 protein of ribonucleotide reductase (Nordlund et al, 1990). The interface between the a and P chains is extensive, burying 4,068 A* of the solvent-exposed surface of each chain.…”

Section: Overall Structurementioning

confidence: 70%

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

et al. 1997

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Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the 02-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type 11, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type I1 methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type I1 Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A and 2.7 8, resolution, respectively, both determined at 18 "C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H20), as well as both carboxylate oxygens of Glu a144. This bis-yhydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 "C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the p chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including Da209E, a ligand of one of the irons.

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Section: Overall Structurementioning

confidence: 70%

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

et al. 1997

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“…Previous studies of inhibitors to protein R2 of the one-electron reduction type have suggested at least three possible mechanisms for inhibitor activity: (i) direct accessibility of the inhibitor to the iron/radical site, perhaps mediated by protein conformational flexibility, (ii) long range electron transfer from an interaction site on or close to the protein surface, or (iii) existence of another more accessible free radical in equilibrium with the tyrosyl radical [7,20,21]. At present it is not possible to unambigously distinguish a common dominating mechanism for the different R2 proteins that have been studied.…”

Section: Discussionmentioning

confidence: 99%

“…coli R2 (2.2 A resolution) has shown that the tyrosyl radical/ iron site is deeply buried inside the protein, about 10 ~ distant from the nearest protein surface, and is surrounded by several hydrophobic amino acid residues [7].…”

Section: Introductionmentioning

confidence: 99%

Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p‐alkoxyphenols

et al. 1995

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The rate of reduction of the tyrosyl radical in the small subunit of ribonucleotide reductase (protein R2) from E. coli, mouse, and herpes simplex virus (HSV-2) by a series of p-alkoxyphenols with different alkyl chains, have been studied by stopped-flow UV-vis and stopped-flow EPR spectroscopy. The reduction and release of iron in R2 by the inhibitors was followed using bathophenanthroline as chelator of Fe 2+. p-Alkoxyphenols reduce the mouse R2 tyrosyl radical 1-2 orders of magnitude faster than the HSV-2 and E. coli radical. In contrast to E. coli, the iron center in R2 from mouse and HSV-2 is reduced by the inhibitors. For mouse R2, the rate of reduction of the tyrosyl radical increases in parallel with increasing alkyl chain length of the inhibitor, an observation which may be important for the design of new antiproliferative drugs.

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“…16 Residues (bold letters) are invariant in all R2 proteins sequenced so far (Asp139 is a Glu in the Epstein-Barr virus protein). Most of them are iron ligands (#), form the hydrophobic pocket (+) around the tyrosyl radical (*) or are proposed to be involved in the electron transfer mechanism (13).…”

Section: Expression Of 22 B Brucei Nrd B In E Colimentioning

confidence: 99%

“…1). Asp85, Glu116, His119, Glu178, Glu212 and His215 correspond to the iron ligands in mouse and E. coli R2 [7,13,14].…”

Section: Sequence Comparison Of 72 Brucei R2 With Other R2 Proteinsmentioning

confidence: 99%

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

et al. 1997

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As a first step towards the understanding of the trypanosomal synthesis of deoxyribonucleotides we describe the cloning of the gene of ribonucleotide reductase subunit R2 from 72 brucei hrucei and its overexpression in E. coli. Materials and methods DNA and RNA preparation2;4 l08 long slender 72 brucei cells were suspended in 10 mM Tris-HC1, 250 mM NaCI, 0.5% Nonidet P-40, pH 8.0. After 5 rain incubation on ice the suspension was centrifuged and the cell pellet was homogenized in 200 lal 10 mM Tris-HC1, 10 mM NaC1, 10 mM EDTA, 0.5% SDS, pH 8.0. 50 lag proteinase K were added and the mixture was incubated for 1 h at 37°C. DNA was purified by phenol extraction.RNA was isolated from bloodstream long slender and short stumpy as well as procyclic stages of 72 brucei using the RNA Easy Kit (Qiagen) according to the instructions of the manufacturer.

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Three-dimensional structure of the free radical protein of ribonucleotide reductase

Cited by 851 publications

References 54 publications

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

Crystal structure of the hydroxylase component of methane monooxygenase fromMethylosinus trichosporiumOB3b

Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p‐alkoxyphenols

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

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