- Formal reduction potential of 3,5-difluorotyrosine in a structured protein: Insight into multistep radical transfer
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The reversible Y-O?/Y-OH redox properties of the α3Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated
- Ravichandran, Kanchana R.,Liang, Li,Stubbe, Joanne,Tommos, Cecilia
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- Cleavage of a carbon–fluorine bond by an engineered cysteine dioxygenase
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Cysteine dioxygenase (CDO) plays an essential role in sulfur metabolism by regulating homeostatic levels of cysteine. Human CDO contains a post-translationally generated Cys93–Tyr157 cross-linked cofactor. Here, we investigated this Cys–Tyr cross-linking by incorporating unnatural tyrosines in place of Tyr157 via a genetic method. The catalytically active variants were obtained with a thioether bond between Cys93 and the halogen-substituted Tyr157, and we determined the crystal structures of both wild-type and engineered CDO variants in the purely uncross-linked form and with a mature cofactor. Along with mass spectrometry and 19F NMR, these data indicated that the enzyme could catalyze oxidative C–F or C–Cl bond cleavage, resulting in a substantial conformational change of both Cys93 and Tyr157 during cofactor assembly. These findings provide insights into the mechanism of Cys–Tyr cofactor biogenesis and may aid the development of bioinspired aromatic carbon–halogen bond activation.
- Li, Jiasong,Griffith, Wendell P.,Davis, Ian,Shin, Inchul,Wang, Jiangyun,Li, Fahui,Wang, Yifan,Wherritt, Daniel J.,Liu, Aimin
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- Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping
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Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and t
- Yee, Estella F.,Dzikovski, Boris,Crane, Brian R.
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supporting information
p. 17571 - 17587
(2019/11/05)
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- Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa on the Forward and Reverse Ground-State Reactions
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The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the pKa of Y21 increases the rate of dark state recovery by 4000-fold with a Br?nsted coefficient of ~1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of ~6-8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications.
- Gil, Agnieszka A.,Haigney, Allison,Laptenok, Sergey P.,Brust, Richard,Lukacs, Andras,Iuliano, James N.,Jeng, Jessica,Melief, Eduard H.,Zhao, Rui-Kun,Yoon, EunBin,Clark, Ian P.,Towrie, Michael,Greetham, Gregory M.,Ng, Annabelle,Truglio, James J.,French, Jarrod B.,Meech, Stephen R.,Tonge, Peter J.
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p. 926 - 935
(2016/02/05)
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- Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs
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While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.
- Yu, Yang,Lv, Xiaoxuan,Li, Jiasong,Zhou, Qing,Cui, Chang,Hosseinzadeh, Parisa,Mukherjee, Arnab,Nilges, Mark J.,Wang, Jiangyun,Lu, Yi
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supporting information
p. 4594 - 4597
(2015/04/27)
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- Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase
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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 (FnYs, n = 1-4) are promising tools for studying the mechanism of Y? formation and reactivity, as their pK 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-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs 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 FnY(s), and the resulting proteins are isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y? in wild-type RNR, generate long-lived FnY?s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-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-F3Y? facilitate spectral subtractions that make detection and identification of new Y?s straightforward.
- Minnihan, Ellen C.,Young, Douglas D.,Schultz, Peter G.,Stubbe, Joanne
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supporting information; experimental part
p. 15942 - 15945
(2011/11/13)
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- Synthesis of 3,5-difluorotyrosine-containing peptides: Application in substrate profiling of protein tyrosine phosphatases
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(Chemical Equation Presented) Fully protected 3,5-difluorotyrosine (F 2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The Fsub
- Gopishetty, Bhaskar,Ren, Lige,Waller, Tiffany M.,Wavreille, Anne-Sophie,Lopez, Miguel,Thakkar, Amit,Zhu, Jinge,Pei, Dehua
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supporting information; experimental part
p. 4605 - 4608
(2009/05/30)
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- Mono-, di-, tri-, and tetra-substituted fluorotyrosines: New probes for enzymes that use tyrosyl radicals in catalysis
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A set of N-acylated, carboxyamide fluorotyrosine (FnY) analogues [Ac-S-FY-NH2, Ac-3,5-F2Y-NH2, Ac-2,3-F 2Y-NH2, Ac-2,3,5-F3Y-NH2, Ac-2,3,6-F3Y-NH2 and Ac-2,3,5,6-F4Y-NH 2] have been synthesized from their corresponding amino acids to interrogate the detailed reaction mechanism(s) accessible to F nY?s in small molecules and in proteins. These Ac-F nY-NH2 derivatives span a pKa range from 5.6 to 8.4 and a reduction potential range of 320 mV in the pH region accessible to most proteins (6-9). DFT electronic-structure calculations capture the observed trends for both the reduction potentials and pKaS. Dipeptides of the methyl ester of 4-benzoyl-L-phenylalanyl-FnYs at pH 4 were examined with a nanosecond laser pulse and transient absorption spectroscopy to provide absorption spectra of FnY?s. The EPR spectrum of each F nY? has also been determined by UV photolysis of solutions at pH 11 and 77 K. The ability to vary systematically both pKa and radical reduction potential, together with the facility to monitor radical formation with distinct absorption and EPR features, establishes that FnYs will be useful in the study of biological charge-transport mechanisms involving tyrosine. To demonstrate the efficacy of the fluorotyrosine method in unraveling charge transport in complex biological systems, we report the global substitution of tyrosine by 3-fluorotyrosine (3-FY) in the R2 subunit of ribonucleotide reductase (RNR) and present the EPR spectrum along with its simulation of 3-FY122?. In the companion paper, we demonstrate the utility of FnYs in providing insight into the mechanism of tyrosine oxidation in biological systems by incorporating them site-specifically at position 356 in the R2 subunit of Escherichia coli RNR.
- Seyedsayamdost, Mohammad R.,Reece, Steven Y.,Nocera, Daniel G.,Stubbe, JoAnne
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p. 1569 - 1579
(2007/10/03)
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- Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions
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Protein tyrosine kinases catalyze the transfer of the γ-phosphoryl group from ATP to tyrosine residues in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of fluorotyrosine-containing peptide substrates. It was established for five such fluorotyrosine-containing peptide substrates that there is good agreement between the tyrosine analogue phenol pK(a) and the ionizable group responsible for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol must be neutral to be enzymatically active. Taken together with previous data indicating a small β(nucleophile) coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the β(leaving group) coefficient was measured for the reverse protein tyrosine kinase reaction and shown to be -0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state with significant proton transfer to the departing phenol.
- Kim, Kyonghee,Cole, Philip A.
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p. 6851 - 6858
(2007/10/03)
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