<i>Bacillus subtilis</i> Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

The class Ib ribonucleotide reductase (RNR) isolated from <i>Bacillus subtilis</i> was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn^III₂-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn^III₂-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min^–1 mg^–1). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that <i>B. subtilis</i> RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of <i>B. subtilis</i> RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn^III₂-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β₂. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ∼1250 nmol min^–1 mg^–1 using a 1:1 mixture of NrdE:Mn^III₂-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (∼1 μM), NrdE is a monomer (α) and Mn^III₂-Y· NrdF is a dimer (β₂). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations

Dph3 Is an Electron Donor for Dph1-Dph2 in the First Step of Eukaryotic Diphthamide Biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. The biosynthesis of diphthamide was proposed to involve three steps. The first step is the transfer of the 3-amino-3-carboxypropyl group from <i>S</i>-adenosyl-l-methionine (SAM) to the histidine residue of EF2, forming a C–C bond. Previous genetic studies showed this step requires four proteins in eukaryotes, Dph1–Dph4. However, the exact molecular functions for the four proteins are unknown. Previous study showed that Pyrococcus horikoshii Dph2 (PhDph2), a novel iron-sulfur cluster-containing enzyme, forms a homodimer and is sufficient for the first step of diphthamide biosynthesis <i>in vitro</i>. Here we demonstrate by <i>in vitro</i> reconstitution that yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step <i>in vitro</i> in the presence of dithionite as the reductant. We further demonstrate that yeast Dph3 (also known as KTI11), a CSL-type zinc finger protein, can bind iron and in the reduced state can serve as an electron donor to reduce the Fe-S cluster in Dph1-Dph2. Our study thus firmly establishes the functions for three of the proteins involved in eukaryotic diphthamide biosynthesis. For most radical SAM enzymes in bacteria, flavodoxins and flavodoxin reductases are believed to serve as electron donors for the Fe-S clusters. The finding that Dph3 is an electron donor for the Fe-S clusters in Dph1-Dph2 is thus interesting and opens up new avenues of research on electron transfer to Fe-S proteins in eukaryotic cells