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

Abstract

Here, 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 radicaldependent 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, Fe2+, and O2 to produce ~1 Y·/β2 and ~3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’s (6.4 to 7.8) and reduction potentialsmore » (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry.« less

Authors:
 [1]; ; ;  [1];  [1];  [2];  [1];
  1. Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States
  2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
Publication Date:
Research Org.:
Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1259739
Alternate Identifier(s):
OSTI ID: 1260961
Grant/Contract Number:  
FG02-11ER16282
Resource Type:
Published Article
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Name: Journal of the American Chemical Society Journal Volume: 138 Journal Issue: 25; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Oyala, Paul H., Ravichandran, Kanchana R., Funk, Michael A., Stucky, Paul A., Stich, Troy A., Drennan, Catherine L., Britt, R. David, and Stubbe, JoAnne. Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example. United States: N. p., 2016. Web. https://doi.org/10.1021/jacs.6b03605.
Oyala, Paul H., Ravichandran, Kanchana R., Funk, Michael A., Stucky, Paul A., Stich, Troy A., Drennan, Catherine L., Britt, R. David, & Stubbe, JoAnne. Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example. United States. https://doi.org/10.1021/jacs.6b03605
Oyala, Paul H., Ravichandran, Kanchana R., Funk, Michael A., Stucky, Paul A., Stich, Troy A., Drennan, Catherine L., Britt, R. David, and Stubbe, JoAnne. Tue . "Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example". United States. https://doi.org/10.1021/jacs.6b03605.
@article{osti_1259739,
title = {Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example},
author = {Oyala, Paul H. and Ravichandran, Kanchana R. and Funk, Michael A. and Stucky, Paul A. and Stich, Troy A. and Drennan, Catherine L. and Britt, R. David and Stubbe, JoAnne},
abstractNote = {Here, 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 radicaldependent 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, Fe2+, and O2 to produce ~1 Y·/β2 and ~3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’s (6.4 to 7.8) and reduction potentials (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry.},
doi = {10.1021/jacs.6b03605},
journal = {Journal of the American Chemical Society},
number = 25,
volume = 138,
place = {United States},
year = {2016},
month = {6}
}

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