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PDBsum entry 5ci3
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Oxidoreductase
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PDB id
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5ci3
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References listed in PDB file
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Key reference
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Title
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Biophysical characterization of fluorotyrosine probes site-Specifically incorporated into enzymes: e. Coli ribonucleotide reductase as an example.
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Authors
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P.H.Oyala,
K.R.Ravichandran,
M.A.Funk,
P.A.Stucky,
T.A.Stich,
C.L.Drennan,
R.D.Britt,
J.Stubbe.
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Ref.
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J Am Chem Soc, 2016,
138,
7951-7964.
[DOI no: ]
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PubMed id
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Abstract
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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 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 (19)F 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.
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