Diversity in Overall Activity Regulation of Ribonucleotide Reductase

Venkateswara Rao Jonna,Mikael Crona,Reza Rofougaran,Daniel Lundin,Samuel Johansson,Kristoffer Brännström,Britt-Marie Sjöberg,Anders Hofer

doi:10.1074/jbc.m115.649624

Venkateswara Rao Jonna, Mikael Crona + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m115.649624

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Abstract

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.

Highlights

Ribonucleotide reductase (RNR) makes DNA building blocks
P. aeruginosa Class I RNR Belongs to a Subclass with Varying Numbers of ATP Cones—A bioinformatics analysis was performed to determine how common multiple ATP cones are in RNRs
We first searched all unique RNR sequences found in the non-redundant database of NCBI with the Pfam ATP cone domain (PF03477) to determine the number of ATP cones in the three RNR classes as well as in the new subclasses proposed in RNRdb2 (Table 1)

Summary

Background

Ribonucleotide reductase (RNR) makes DNA building blocks. Results: Binding of three dATP molecules to the Pseudomonas aeruginosa class I RNR ␣ subunit inactivates the enzyme by inducing an inert ␣4 complex. There are similarities, and clear differences, in action between the bacterial E. coli RNR and the eukaryotic enzymes In both mechanisms, high dNTP levels mediate oligomerization into tight complexes of larger size than the common ␣2␤2 complex, and the ␤2 and ␣2n subunits can no longer interact in a productive way. In the eukaryotic class I enzymes, the overall activity regulation relies on two different types of ␣6 complexes depending on whether dATP or ATP binds to the a-site [8, 9]. We show that RNRs with multiple ATP cones are much more common among RNRs than previously realized and that the P. aeruginosa class I RNR differs in several aspects from both the eukaryotic and the E. coli mechanistic models of overall activity regulation. The distinct differences in quaternary structures number of ATP cones among RNRs suggest that the mechanism of activity regulation might form the basis for the development of novel antibiotics

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