Mutant R1 Proteins from Escherichia coli Class Ia Ribonucleotide Reductase with Altered Responses to dATP Inhibition

Abstract

Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.A key enzyme of nucleic acid metabolism in the cell is ribonucleotide reductase. This enzyme, abbreviated RNR, 1 catalyzes the reaction where ribonucleotides are converted to deoxyribonucleotides, thus providing the cell with all components of DNA. RNR activity is essential for DNA synthesis and repair, but is also in need of a strict control system. An uncontrolled production of DNA precursors is devastating to the cell since the mutation frequency is increased at aberrant concentrations of dNTPs (1). In prokaryotic as well as most eukaryotic RNRs belonging to the class Ia enzymes, this is solved by an allosteric regulation controlling both the substrate specificity and the overall activity. Other classes of RNR, denoted Ib, II, and III, all perform the same reaction but operate at different conditions and via slightly different catalytic mechanisms (for a review, see Ref.2). Class Ib and II RNRs are allosterically regulated in a similar manner, and the class III enzymes are regulated approximately by the same effectors as class I RNRs (Fig. 1). In this study, all studies concern the class Ia RNR from Escherichia coli.The E. coli class Ia enzyme is composed of two dimeric proteins with different properties. The larger protein, denoted R1, contains the catalytic site and two types of allosteric sites. The catalytic mechanism involves advanced chemistry that is accomplished by a stable amino acid radical that originates from the smaller subunit, denoted R2. The fre...

Enhancement by Effectors and Substrate Nucleotides of R1-R2 Interactions in Escherichia coli Class Ia Ribonucleotide Reductase

“…This increase of R1-R2 affinity coincides with the inhibition of enzyme activity (Ref. 23 and this study). Even though our results show increasing amounts of R1 bound to R2 when the interaction becomes tighter, similarly high stoichiometries were also obtained in the presence of positive effectors (Table III) and in the active complex (Table IV).…”

“…Most intriguing is the effect of dATP, which is a positive allosteric effector, when bound to the specificity site where dTTP, dGTP, and ATP also bind but impose negative allosteric effects on enzyme activity when bound to the overall activity site (where only ATP can also bind). The K D values for dATP bound to the specificity and the overall activity sites of protein R1 are 0.9 and 6.3 M, respectively (23), and the apparent K L values for positive and negative allosteric effects on enzyme activity are 0.2 and 1.7 M, respectively (23). Therefore, the R1-R2 interaction on the Biacore membrane was studied at a dATP concentration between 1 M and 1 mM.…”

“…This methodology has been used recently to study the strength of complexes formed between wild type E. coli R2 protein and E. coli R1 proteins with mutated overall activity sites (23). Plasmid pTB2 (8) containing the nrdB gene coding for protein R2 was used as template for insertion of hexahistidine tags in R2.…”

Functional Analysis of the Streptomyces coelicolor NrdR ATP-Cone Domain: Role in Nucleotide Binding, Oligomerization, and DNA Interactions

“…The crystal structure of the E. coli NrdA dimer with bound nucleotide shows that the ATP-cone domain is located at the N-terminal tip, at the surface of the molecule (14,48). Two H-bonded histidines, His59 and His88, located at and near the nucleotide binding site, respectively, participate in forming the dimer contact region and in communicating allosteric inhibition (4). The overall activity site occupied by the ligand is open to the solvent and favorably disposed to allow subunit interactions.…”

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

“…The activity site is located at the N terminus of the R1 subunit and functions as an "on-off" switch: ATP binding leads to an active enzyme, whereas dATP binding inhibits the enzyme. Hence, by monitoring the ATP/dATP ratio, the enzyme aims to ensure an overall dNTP level that is presumably optimal for DNA replication (1,8).…”