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A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

ABSTRACT: Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit ? is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-? assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger ?-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of ? with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-?, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of ? self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

SUBMITTER: Fu Y

PROVIDER: S-EPMC4271543 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

Fu Yuan Y Lin Hongyu H Wisitpitthaya Somsinee S Blessing William A WA Aye Yimon Y

Chembiochem : a European journal of chemical biology 20140924 17

Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically t ...[more]

PMID: 25256246

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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

A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

Publications

A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

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