Novel ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit

Abstract

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.

A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant

“…F. ignava RNR instead carries an ATP cone in its NrdB protein, between the N-terminal Grx domain and the radical-generating domain. We have recently reported a similar N-terminal ATP-cone fusion to NrdB in Leeuwenhoekiella blandensis ( 8 ). Both of these fusion proteins belong to the NrdBi subclass, which harbors a few additional ATP-cone::NrdB fusions.…”

“…Currently, class I RNRs have been subclassified based on radical cofactor type (subclasses Ia, Ib, Ic, Id, and Ie) or evolutionary history (subclasses NrdA/B followed by a small letter plus subclass NrdE/F) ( 1 , 6 ). Metal content does not always follow phylogeny because two unrelated Mn 2 subclasses exist, where one subclass contains a tyrosyl radical in the vicinity of a Mn III /Mn III center (Ib, NrdE/F), and another recently identified subclass (Id, NrdAi/Bi) contains a mixed valent Mn III /Mn IV metal center that harbors the unpaired electron ( 7 – 9 ). In eukaryotic RNRs and several evolutionarily unrelated bacterial class I subclasses, the NrdB component contains a stable tyrosyl radical in the vicinity of a diferric metal center (Ia).…”

“…In short, the enzyme is active when ATP is bound and when dATP is bound, the enzyme is turned off. We have recently shown that the ATP cone can be horizontally transferred between different RNRs and even to different subunits of the holoenzyme ( 8 , 23 ). In an overwhelming number of cases, the ATP cone is an N-terminal domain of the catalytic subunit of RNR ( 23 ).…”

The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron

“…The strictly aerobic class I RNRs are comprised of two homodimeric subunits: a large catalytic subunit R1, where ribonucleotide reduction takes place, and a small radical-generating subunit R2. Allosteric regulation is predominantly performed in the R1 subunit but is also identified in some R2 proteins [10]. Class I R2 proteins are further subdivided into five subgroups (R2a-R2e), based on the radical species and/or metallo-cofactor employed for radical generation (Fig.…”

“…In R2c and R2d subclasses, on the other hand, the radical equivalent is housed on the metal cofactor itself. R2c proteins generate a Mn IV /Fe III cofactor [17][18][19] and the recently discovered class R2d-a Mn IV /Mn III cofactor [10,20,21]. These dimetal centers reside in a ferritin-like scaffold, and are coordinated by two histidine residues and four carboxylate ligands.…”