title: Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.
abstract: Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.
authors.affiliation: Department of Molecular Biology & Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Alex
authors.initials: A
authors.lastname: Kasrayan
authors.forename: Pernilla Larsson
authors.initials: PL
authors.lastname: Birgander
authors.forename: Lucia
authors.initials: L
authors.lastname: Pappalardo
authors.forename: Karin
authors.initials: K
authors.lastname: Regnström
authors.forename: MariAnn
authors.initials: M
authors.lastname: Westman
authors.forename: Agneta
authors.initials: A
authors.lastname: Slaby
authors.forename: Euan
authors.initials: E
authors.lastname: Gordon
authors.forename: Britt-Marie
authors.initials: BM
authors.lastname: Sjöberg
mesh_terms: D000494:Allosteric Regulation; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides; D010084:Oxidation-Reduction; D021122:Protein Subunits; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013879:Thioredoxins / Q000494:pharmacology
chemical_list: D009711:Nucleotides; D021122:Protein Subunits; D013879:Thioredoxins; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit