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A Ferredoxin Disulfide Reductase Delivers Electronsto the Methanosarcina barkeri Class III RibonucleotideReductase

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ABSTRACT

Twosubtypes of class III anaerobic ribonucleotide reductases (RNRs)studied so far couple the reduction of ribonucleotides to the oxidationof formate, or the oxidation of NADPH via thioredoxin and thioredoxinreductase. Certain methanogenic archaea contain a phylogeneticallydistinct third subtype of class III RNR, with distinct active-siteresidues. Here we report the cloning and recombinant expression ofthe Methanosarcina barkeri class III RNR and showthat the electrons required for ribonucleotide reduction can be deliveredby a [4Fe-4S] protein ferredoxin disulfide reductase, and a conservedthioredoxin-like protein NrdH present in the RNR operon. The diversityof class III RNRs reflects the diversity of electron carriers usedin anaerobic metabolism.

No MeSH data available.


EPR spectrum of the MbNrdD G• (40 μM NrdDpeptide).
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fig3: EPR spectrum of the MbNrdD G• (40 μM NrdDpeptide).

Mentions: For ourstudies of NrdD3, we chose the model methanogen M. barkeri.43 Initial attempts to purify MbNrdDwere confounded by the instability of the protein, which aggregatedover time and bound tightly to various chromatographic resins andto DNA. We later found that this instability and nonspecific bindingcould be overcome by addition of 200–300 mM KCl and/or 20%glycerol to the buffers used for chromatography, storage, and assays.To generate active MbNrdD for biochemical studies, we incubated MbNrdDwith MbNrdG and SAM in the presence of the diaminoacridine/bicinephotoreduction system,3 resulting in thegeneration of a radical with a characteristic doublet EPR signal (Figure 3), consistent withits assignment as G•. MbNrdD stored in buffer containing20 mM Tris (pH 7.5), 300 mM KCl, and 20% glycerol was stable at 4°C in the glovebox for several weeks, during which a yield of∼0.1 G• per NrdD peptide was reproduciblyobtained.


A Ferredoxin Disulfide Reductase Delivers Electronsto the Methanosarcina barkeri Class III RibonucleotideReductase
EPR spectrum of the MbNrdD G• (40 μM NrdDpeptide).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697749&req=5

fig3: EPR spectrum of the MbNrdD G• (40 μM NrdDpeptide).
Mentions: For ourstudies of NrdD3, we chose the model methanogen M. barkeri.43 Initial attempts to purify MbNrdDwere confounded by the instability of the protein, which aggregatedover time and bound tightly to various chromatographic resins andto DNA. We later found that this instability and nonspecific bindingcould be overcome by addition of 200–300 mM KCl and/or 20%glycerol to the buffers used for chromatography, storage, and assays.To generate active MbNrdD for biochemical studies, we incubated MbNrdDwith MbNrdG and SAM in the presence of the diaminoacridine/bicinephotoreduction system,3 resulting in thegeneration of a radical with a characteristic doublet EPR signal (Figure 3), consistent withits assignment as G•. MbNrdD stored in buffer containing20 mM Tris (pH 7.5), 300 mM KCl, and 20% glycerol was stable at 4°C in the glovebox for several weeks, during which a yield of∼0.1 G• per NrdD peptide was reproduciblyobtained.

View Article: PubMed Central - PubMed

ABSTRACT

Twosubtypes of class III anaerobic ribonucleotide reductases (RNRs)studied so far couple the reduction of ribonucleotides to the oxidationof formate, or the oxidation of NADPH via thioredoxin and thioredoxinreductase. Certain methanogenic archaea contain a phylogeneticallydistinct third subtype of class III RNR, with distinct active-siteresidues. Here we report the cloning and recombinant expression ofthe Methanosarcina barkeri class III RNR and showthat the electrons required for ribonucleotide reduction can be deliveredby a [4Fe-4S] protein ferredoxin disulfide reductase, and a conservedthioredoxin-like protein NrdH present in the RNR operon. The diversityof class III RNRs reflects the diversity of electron carriers usedin anaerobic metabolism.

No MeSH data available.