The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Filatov, Dmitri; Ingemarson, Rolf; Gräslund, Astrid; Thelander, Lars

doi:10.1016/s0021-9258(19)49608-7

Cited by 34 publications

(11 citation statements)

References 32 publications

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“…Structural differences among the diferric sites in different R2 proteins are indicated by other spectroscopic studies (Mann et al, 1991;Thelander et al, 1994;Filatov et al, 1992;Sahlin et al, 1987;Lankinen et al, 1982;Ingemarson et al, 1989). The light absorption spectrum of the diferric site in mouse metR2 is characterized by absorption bands at 320 and 370 nm (Figure 6) which are assigned to ligandto-metal charge transfer from O 2to Fe(III).…”

Section: Presence Of An Oxo Bridge In the Primary Mixed-valentmentioning

confidence: 73%

“…Recombinant mouse and HSV1 R2 proteins were prepared, reacted with iron/ascorbate, and desalted as previously reported (Mann et al, 1991). A truncated form of the HSV1 protein R2 lacking the seven carboxyl-terminal amino acid residues GAVVDL was produced according to Filatov et al (1992). Protein metR2 from E. coli was prepared as described in McClarty et al (1980).…”

Section: Methodsmentioning

confidence: 99%

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EPR Study of the Mixed-Valent Diiron Sites in Mouse and Herpes Simplex Virus Ribonucleotide Reductases. Effect of the Tyrosyl Radical on Structure and Reactivity of the Diferric Center

Davydov

Ingemarson

et al. 1997

Biochemistry

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Reduction of ribonucleotide reductase (EC 1.17.4.1) R2 proteins in a frozen glycerol-buffer solution at 77 K by mobile electrons generated by gamma-irradiation produces EPR-detectable iron sites in mixed-valent Fe(II)/Fe(III) states. The primary EPR signals give information about the ligand arrangement of the diferric form of the iron site, whereas secondary signals observed after annealing of the sample show the effects of structural relaxation. In recombinant metR2 proteins (without free radical) from mouse and herpes virus type 1, the mixed-valent sites trapped at 77 K give rise to axial S = 1/2 EPR spectra with g values in the range 1.79-1.94, observable at temperatures up to 110 K. The spectra are assigned to mu-oxo-bridged dinuclear iron sites. In mouse metR2, the primary EPR spectrum is a mixture of two components. Annealing the R2 samples to 160-170 K transforms the primary EPR signals into rhombic spectra, characterized by gav < 1.8, and observable only below 25 K. These spectra are assigned to partially relaxed forms with a mu-hydroxo bridge, formed by protonation of the oxo bridge. Further annealing at 220 K produces new rhombic EPR spectra, which are closely similar with those observed and found to be stable after chemical reduction at room temperature. The EPR signal of the primary mixed-valent iron site in active mouse R2 protein with a tyrosyl radical also has two components. Both are different from those observed in metR2. In herpes simplex virus type 1 protein R2, one primary mixed-valent component was observed for the met protein. The dose-yield curve for the mixed-valent state in active mouse R2 is sigmoidal in shape, indicating that the tyrosyl radical is reduced by mobile electrons before the iron site. Kinetic experiments on the reduction by dithionite on mouse R2 without and with radical show a significantly enhanced rate for reduction of the iron site in the protein without radical. The results suggest that in active mouse R2 only complete diferric sites with neighboring radicals give rise to the mixed-valent spectra, and that these sites may exist in two structurally distinct forms. The results on the mouse R2 proteins confirm and extend previous results obtained on the Escherichia coli protein R2 showing that the presence of the tyrosyl radical significantly affects not only the structure but also the reactivity of the iron site.

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Section: Presence Of An Oxo Bridge In the Primary Mixed-valentmentioning

confidence: 73%

Section: Methodsmentioning

confidence: 99%

EPR Study of the Mixed-Valent Diiron Sites in Mouse and Herpes Simplex Virus Ribonucleotide Reductases. Effect of the Tyrosyl Radical on Structure and Reactivity of the Diferric Center

Davydov

Ingemarson

et al. 1997

Biochemistry

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“…It is still unclear which residue in the R2 protein could link this chain to Trp48. According to site-directed mutagenesis of the R2 protein, a conserved tyrosine (Tyr356) is very important for enzyme activity (Climent et al, 1992;Filatov et al, 1992). It is possible that this tyrosine is the linking residue, but due to the very flexible C-terminal region of E. coli R2, this residue could not be localized in the 3-D structure (Nordlund et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Evidence by Site-Directed Mutagenesis Supports Long-Range Electron Transfer in Mouse Ribonucleotide Reductase

Rova¹,

Goodtzova²,

Ingemarson³

et al. 1995

Biochemistry

116

120

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Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

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“…Our inhibitors act as mimics of the C-terminus of the HSV RR small subunit (R2). This C-terminal region binds to the RR large subunit (R1) thus enabling subunit association and subsequent catalytic activity . Inhibition results from competitive binding of the inhibitors to R1 which prevents formation of the catalytically active holoenzyme.…”

Section: Introductionmentioning

confidence: 99%

“…This C-terminal region binds to the RR large subunit (R1) thus enabling subunit association and subsequent catalytic activity. 2 Inhibition results from competitive binding of the inhibitors to R1 which prevents formation of the catalytically active holoenzyme. An attractive characteristic of this type of inhibition is that compounds based on the HSV R2 C-terminal sequence are selective for HSV RR over human RR.…”

Section: Introductionmentioning

confidence: 99%

Ureido-Based Peptidomimetic Inhibitors of Herpes Simplex Virus Ribonucleotide Reductase: An Investigation of Inhibitor Bioactive Conformation

et al. 1996

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We have been investigating peptidomimetic inhibitors of herpes simplex virus (HSV) ribonucleotide reductase (RR). These inhibitors bind to the HSV RR large subunit and consequently prevent subunit association and subsequent enzymatic activity. This report introduces a new series of compounds that contain an extra nitrogen (a ureido function) at the inhibitor N-terminus. This nitrogen improves inhibitor binding potency 50-fold over our first published inhibitor series. Evidence supports that this improvement in potency results from a new hydrogen-bonding contact between the inhibitor and the RR large subunit. This report also provides evidence for the bioactive conformation around two important amino acid residues contained in our inhibitors. A tert-butyl group, which contributes 100-fold to inhibitor potency but does not directly bind to the large subunit, favors an extended beta-strand conformation that is prevalent in solution and in the bound state. More significantly, the bioactive conformation around a pyrrolidine-modified asparagine residue, which contributes over 30 000-fold to inhibitor potency, is elucidated through a series of conformationally restricted analogues.

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The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Cited by 34 publications

References 32 publications

EPR Study of the Mixed-Valent Diiron Sites in Mouse and Herpes Simplex Virus Ribonucleotide Reductases. Effect of the Tyrosyl Radical on Structure and Reactivity of the Diferric Center

EPR Study of the Mixed-Valent Diiron Sites in Mouse and Herpes Simplex Virus Ribonucleotide Reductases. Effect of the Tyrosyl Radical on Structure and Reactivity of the Diferric Center

Evidence by Site-Directed Mutagenesis Supports Long-Range Electron Transfer in Mouse Ribonucleotide Reductase

Ureido-Based Peptidomimetic Inhibitors of Herpes Simplex Virus Ribonucleotide Reductase: An Investigation of Inhibitor Bioactive Conformation

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