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 protein­(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 (Y₁₂₂•, <i>Escherichia coli</i>) in subunit β of a cysteine (C₄₃₉) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y₁₂₂ ⇆ Y₃₅₆ in β to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ 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 Y₃₅₆-β, Y₇₃₁- and Y₇₃₀-α, and each protein was incubated with the appropriate second subunit β­(α), CDP and effector ATP to trap an amino tyrosyl radical (NH₂Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH₂Y•s reported the <i>g</i>_<i>x</i> values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. ²H 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 NH₂Y₇₃₀•, one to NH₂Y₇₃₁• and none to NH₂Y₃₅₆•. Similar experiments with double mutants α-NH₂Y₇₃₀/C₄₃₉A and α-NH₂Y₇₃₁/Y₇₃₀F 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 (NH₂Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH₂Y of key importance for its application in mechanistic studies. By combining the tRNA/NH₂Y-RS suppression technology with a model protein tailored for amino acid redox studies (α₃X, X = NH₂Y), the formal reduction potential of NH₂Y₃₂(O^•/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 NH₂Y₃₂(O^•/OH) and Y₃₂(O^•/OH) when measured under reversible conditions is ∼300–400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH₂Y and Y. We have also generated D₆-NH₂Y₇₃₁-α2 of ribonucleotide reductase (RNR), which when incubated with β2/CDP/ATP generates the D₆-NH₂Y₇₃₁^•-α2/β2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz ¹H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH₂^• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH₂Y-RNR incorporated by infidelity of the tRNA/NH₂Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH₂Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH₂Y-substituted RNRs