Incorporation of Fluorotyrosines into Ribonucleotide Reductase Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase
Author(s)Young, Douglas D.; Schultz, Peter G.; Stubbe, JoAnne; Minnihan, Ellen Catherine
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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 (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.
DepartmentMassachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry
Journal of the American Chemical Society
American Chemical Society (ACS)
Minnihan, Ellen C., Douglas D. Young, Peter G. Schultz, and JoAnne Stubbe. “Incorporation of Fluorotyrosines into Ribonucleotide Reductase Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase.” Journal of the American Chemical Society 133, no. 40 (October 12, 2011): 15942-15945.
Author's final manuscript