2 research outputs found
<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<sup>III</sup><sub>2</sub>-Y·)
cofactor. The activity of this RNR and the one reconstituted from
recombinantly expressed NrdE and reconstituted Mn<sup>III</sup><sub>2</sub>-Y· NrdF using dithiothreitol as the reductant, however,
was low (160 nmol min<sup>–1</sup> mg<sup>–1</sup>).
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<sup>III</sup><sub>2</sub>-Y· NrdF, yielding enzyme containing
4 Mn and 1 Y·/β<sub>2</sub>. 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<sup>–1</sup> mg<sup>–1</sup> using a 1:1 mixture of NrdE:Mn<sup>III</sup><sub>2</sub>-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<sup>III</sup><sub>2</sub>-Y· NrdF is a dimer
(β<sub>2</sub>). 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