3 research outputs found
YBR246W Is Required for the Third Step of Diphthamide Biosynthesis
Diphthamide, the target of diphtheria toxin, is a post-translationally
modified histidine residue that is found in archaeal and eukaryotic
translation elongation factor 2. The biosynthesis and function of
this modification has attracted the interest of many biochemists for
decades. The biosynthesis has been known to proceed in three steps.
Proteins required for the first and second steps have been identified,
but the proteinÂ(s) required for the last step have remained elusive.
Here we demonstrate that the YBR246W gene in yeast is required for
the last step of diphthamide biosynthesis, as the deletion of YBR246W
leads to the accumulation of diphthine, which is the enzymatic product
of the second step of the biosynthesis. This discovery will provide
important information leading to the complete elucidation of the full
biosynthesis pathway of diphthamide
Hydrogen Bond Network between Amino Acid Radical Intermediates on the Proton-Coupled Electron Transfer Pathway of <i>E. coli</i> α2 Ribonucleotide Reductase
Ribonucleotide
reductases (RNRs) catalyze the conversion of ribonucleotides
to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation
of nucleotide diphosphate (NDP) reduction requires a reversible oxidation
over 35 Å by a tyrosyl radical (Y<sub>122</sub>•, <i>Escherichia coli</i>) in subunit
β of a cysteine (C<sub>439</sub>) in the active site of subunit
α. This radical transfer (RT) occurs by a specific pathway involving
redox active tyrosines (Y<sub>122</sub> ⇆ Y<sub>356</sub> in
β to Y<sub>731</sub> ⇆ Y<sub>730</sub> ⇆ C<sub>439</sub> in α); each oxidation necessitates loss of a proton
coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine
was site-specifically incorporated in place of Y<sub>356</sub>-β,
Y<sub>731</sub>- and Y<sub>730</sub>-α, and each protein was
incubated with the appropriate second subunit βÂ(α), CDP
and effector ATP to trap an amino tyrosyl radical (NH<sub>2</sub>Y•)
in the active α2β2 complex. High-frequency (263 GHz) pulse
electron paramagnetic resonance (EPR) of the NH<sub>2</sub>Y•s
reported the <i>g</i><sub><i>x</i></sub> values
with unprecedented resolution and revealed strong electrostatic effects
caused by the protein environment. <sup>2</sup>H electron–nuclear
double resonance (ENDOR) spectroscopy accompanied by quantum chemical
calculations provided spectroscopic evidence for hydrogen bond interactions
at the radical sites, i.e., two exchangeable H bonds to NH<sub>2</sub>Y<sub>730</sub>•, one to NH<sub>2</sub>Y<sub>731</sub>•
and none to NH<sub>2</sub>Y<sub>356</sub>•. Similar experiments
with double mutants α-NH<sub>2</sub>Y<sub>730</sub>/C<sub>439</sub>A and α-NH<sub>2</sub>Y<sub>731</sub>/Y<sub>730</sub>F allowed
assignment of the H bonding partner(s) to a pathway residue(s) providing
direct evidence for colinear PCET within α. The implications
of these observations for the PCET process within α and at the
interface are discussed
Properties of Site-Specifically Incorporated 3‑Aminotyrosine in Proteins To Study Redox-Active Tyrosines: <i>Escherichia coli</i> Ribonucleotide Reductase as a Paradigm
3-Aminotyrosine
(NH<sub>2</sub>Y) has been a useful probe to study
the role of redox active tyrosines in enzymes. This report describes
properties of NH<sub>2</sub>Y of key importance for its application
in mechanistic studies. By combining the tRNA/NH<sub>2</sub>Y-RS suppression
technology with a model protein tailored for amino acid redox studies
(α<sub>3</sub>X, X = NH<sub>2</sub>Y), the formal reduction
potential of NH<sub>2</sub>Y<sub>32</sub>(O<sup>•</sup>/OH)
(<i><i>E</i>°′</i> = 395 ± 7
mV at pH 7.08 ± 0.05) could be determined using protein film
voltammetry. We find that the Δ<i><i>E</i>°′</i> between NH<sub>2</sub>Y<sub>32</sub>(O<sup>•</sup>/OH) and
Y<sub>32</sub>(O<sup>•</sup>/OH) when measured under reversible
conditions is ∼300–400 mV larger than earlier estimates
based on irreversible voltammograms obtained on aqueous NH<sub>2</sub>Y and Y. We have also generated D<sub>6</sub>-NH<sub>2</sub>Y<sub>731</sub>-α2 of ribonucleotide reductase (RNR), which when
incubated with β2/CDP/ATP generates the D<sub>6</sub>-NH<sub>2</sub>Y<sub>731</sub><sup>•</sup>-α2/β2 complex.
By multifrequency electron paramagnetic resonance (35, 94, and 263
GHz) and 34 GHz <sup>1</sup>H ENDOR spectroscopies, we determined
the hyperfine coupling (hfc) constants of the amino protons that establish
RNH<sub>2</sub><sup>•</sup> planarity and thus minimal perturbation
of the reduction potential by the protein environment. The amount
of Y in the isolated NH<sub>2</sub>Y-RNR incorporated by infidelity
of the tRNA/NH<sub>2</sub>Y-RS pair was determined by a generally
useful LC-MS method. This information is essential to the utility
of this NH<sub>2</sub>Y probe to study any protein of interest and
is employed to address our previously reported activity associated
with NH<sub>2</sub>Y-substituted RNRs