Unique ATP - cone - driven allosteric regulation of ribonucleotide 1 reductase via the radical - generating subunit

15 Ribonucleotide reductases (RNRs) are key enzymes in DNA synthesis and repair, with 16 sophisticated allosteric mechanisms controlling both substrate specificity and overall activity. 17 In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the 18 catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, 19 we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the 20 Leewenhoekiella blandensis radical-generating subunit regulates activity via modifications of 21 quaternary structure induced by binding of nucleotides. ATP induces enzymatically 22 competent dimers, whereas dATP induces non-productive tetramers, resulting in different 23 holoenzyme complexes. The tetramer forms solely by interactions between ATP-cones, as 24 evidenced by a 2.45 Å crystal structure. We also present evidence for an MnMn metal 25 center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated 26 by evolutionary capture of the domain by the radical-generating subunit. Our findings present 27 a novel opportunity for dATP-regulation of engineered proteins. 28 29 . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/190033 doi: bioRxiv preprint first posted online Sep. 17, 2017;