Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example.

ABSTRACT: Fluorinated tyrosines (FnY's, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y's, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-β2, Fe(2+), and O2 to produce ∼1 Y·/β2 and ∼3 Fe(3+)/β2. The FnY· are

SUBMITTER: Oyala PH 

PROVIDER: S-EPMC4929525 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature