Unique ATP - cone - driven allosteric regulation of ribonucleotide 1 reductase via the radical - generating subunit 2

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 CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder.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;