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

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journal contribution
posted on 12.10.2011 by Ellen C. Minnihan, Douglas D. Young, Peter G. Schultz, JoAnne Stubbe
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 pKa 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.