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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 ? subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in ? and 731 and 730 in ?) 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-? and the X-ray structures of each resulting ? with a diferric cluster are reported and compared with wt-?2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-?2, Fe(2+), and O2 to produce ?1 Y·/?2 and ?3 Fe(3+)/?2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-?2. Each FnY·-?2 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.

SUBMITTER: Oyala PH

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

REPOSITORIES: biostudies-literature

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

Oyala Paul H PH Ravichandran Kanchana R KR Funk Michael A MA Stucky Paul A PA Stich Troy A TA Drennan Catherine L CL Britt R David RD Stubbe JoAnne J

Journal of the American Chemical Society 20160621 25

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 ...[more]

PMID: 27276098

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Project description:A coenzyme B12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced. Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli. The deduced amino acid sequence shows that the catalytic domain of the B12-dependent enzyme from T. acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E. coli, yeast, mammalian viruses, and man. The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not. Evolutionary bridges created by the T. acidophila sequence and that of a B12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B12-dependent enzyme as well. These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme. Sequence similarities show that the N-terminal domain of the T. acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E. coli, which uses neither B12 nor Fe cofactors. A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E. coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases. A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in "post-genomic" biochemistry.

Dataset Information

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

Publications

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

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