Unnatural amino acids: better than the real things?

Considerable effort has been dedicated to the development of technology for the site-specific incorporation of unnatural amino acids into proteins, with nonsense codon suppression and expressed protein ligation emerging as two of the most promising methods. Recent research advances in which these methods have been applied to study protein function and mechanism are briefly highlighted, and the potential of the methods for efficient, widespread future use in vitro and in vivo is critically evaluated

Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122•) that generates a thiyl radical (C439•) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y•-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s–1). Rapid freeze–quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y•-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356• and 0.5 equiv of dCDP, both at 30 s–1. In the absence of an external reducing system, Y356• reduction occurs concomitant with F3Y reoxidation (0.4 s–1) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s–1) is observed prior to steady-state turnover (1.7 s–1). The [Y356•] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122• is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle

Reversible, Long-Range Radical Transfer in E. coli Class Ia Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDPs or NTPs where N = C, U, G, or A) to 2′-deoxynucleotides (dNDPs or dNTPs)1 and are responsible for controlling the relative ratios and absolute concentrations of cellular dNTP pools. For this reason, RNRs play a major role in ensuring the fidelity of DNA replication and repair. RNRs are found in all organisms and are classified based on the metallocofactor used to initiate catalysis,1 with the class Ia RNRs requiring a diferric-tyrosyl radical (Y•) cofactor. The prototypical class Ia RNR from E. coli, the subject of this account, is composed of two subunits, α2 and β2, and is active as an α2β2 complex, as highlighted in Figure 1. α2 houses the catalytic site for substrate (S) reduction and two allosteric effector (E = ATP, dGTP, TTP, and dATP) binding sites that govern which S is reduced (specificity site) and the overall rate of reduction (activity site). β2 contains the essential diferric-Y• cofactor. This unusually stable Y•, located at position 122, has a t1/2 of 4 days at 4 °C in contrast to the μs lifetimes observed for Y•s in solution. Nucleotide reduction occurs by a complex mechanism involving protein- and substrate-derived radicals, some details of which are summarized in Figure 2.1,3 The stable Y122• transiently oxidizes a cysteine (C439) in the catalytic site to a thiyl radical (S•), which reversibly abstracts a 3′-hydrogen atom (H•) from the NDP. The 3′-nucleotide radical rapidly loses water in the first irreversible step.1,3 The reducing equivalents are provided by two local cysteines (C225 and C462), and the resulting disulfide is re-reduced for subsequent turnovers, ultimately by thioredoxin (TR), thioredoxin reductase (TRR), and NADPH.Chemistry and Chemical Biolog

Redox-Linked Changes to the Hydrogen-Bonding Network of Ribonucleotide Reductase β2

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric–tyrosyl radical (Y122O^•) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O^• reduction mechanism in Escherichia coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O^• was studied using reaction-induced FT-IR spectroscopy and [¹³C]­aspartate-labeled β2. These Y122O^• minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated β2

Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of <i>E. coli</i> Ribonucleotide Reductase by Multifrequency EPR Spectroscopy

<i>E. coli</i> ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y₁₂₂•) within the β2 subunit to a cysteine residue (C₄₃₉) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y₁₂₂ → W₄₈ → Y₃₅₆ in β2, across the subunit interface to Y₇₃₁ → Y₇₃₀ → C₄₃₉ in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y₁₂₂• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH₂Y•). In the current study [¹⁵N]- and [¹⁴N]-NH₂Y₇₃₀• have been generated in H₂O and D₂O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH₂Y₇₃₀•. The <i>g</i> tensor (<i>g</i>_<i>x</i> = 2.0052, <i>g</i>_<i>y</i> = 2.0042, <i>g</i>_<i>z</i> = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH₂ moiety are consistent with an intramolecular hydrogen bond within NH₂Y₇₃₀•. This analysis is an essential first step in using the detailed structure of NH₂Y₇₃₀• to formulate a model for a PCET mechanism within α2 and for use of NH₂Y in other systems where transient Y•s participate in catalysis

Incorporation of Fluorotyrosines into Ribonucleotide Reductase Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase

Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F[subscript n]Ys, n = 1–4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pK[subscript a] values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F[subscript 3]Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F[subscript n]Ys while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F[subscript n]Ys site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F[subscript n]Y(s), and the resulting proteins are isolated in good yields. F[subscript n]Ys incorporated at position 122 of β2, the site of a stable Y· in wild-type RNR, generate long-lived F[subscript n]Y·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y[subscript 122](2,3,5)F[subscript 3]Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y· can accumulate. The distinct EPR properties of the 2,3,5-F[subscript 3]Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward.National Institutes of Health (U.S.) (Grant GM29595

Kinetics of Radical Intermediate Formation and Deoxynucleotide Production in 3-Aminotyrosine-Substituted Escherichia coli

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to 2′-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C[subscript 439]) in α2 by a stable diferric tyrosyl radical (Y[subscript 122]•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y[subscript 122]•→ W[subscript 48]→ Y[subscript 356] in β2 to Y[subscript 731]→ Y[subscript 730]→ C[subscript 439] in α2. To study the details of this process, 3-aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated in place of Y[subscript 356] of β. The resulting protein, Y[subscript 356]NH[subscript 2]Y-β2, and the previously generated proteins Y[subscript 731]NH[subscript 2]Y-α2 and Y[subscript 730]NH[subscript 2]Y-α2 (NH[subscript 2]Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2–0.7 s[superscript –1]. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH[subscript 2]Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH[subscript 2]Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH[subscript 2]Y• formation is biphasic (k[subscript fast] of 9–46 s[superscript –1] and k[subscript slow] of 1.5–5.0 s[subscript –1]) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH[subscript 2]Y• formation, while the k[subscript cat] of 0.5 s[superscript –1] is proposed to be associated with rate-limiting oxidation by NH[subscript 2]Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y[subscript 730]NH[subscript 2]Y-α2 and Y[subscript 731]NH[subscript 2]Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.National Institutes of Health (U.S.) (GM29595