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.
Highlights
Allosteric regulation of an enzyme is defined as regulation of activity by binding of an effector molecule to a different location of the enzyme than the active site
Using a foursubstrate activity assay in the presence of saturating concentrations of the s-site effectors dTTP, dGTP or ATP, we found that L. blandensis Ribonucleotide reductases (RNRs) has a similar specificity regulation pattern to most characterized RNRs (Hofer et al, 2012)
The ATP-cone is only found in some RNRs, and appears to be gained by domain shuffling when evolutionary selection favours it and lost when selection decreases (Lundin et al, 2015)
Summary
Allosteric regulation of an enzyme is defined as regulation of activity by binding of an effector molecule to a different location of the enzyme than the active site. RNRs are essential enzymes in all free-living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors for DNA synthesis and repair (Hofer et al, 2012; Nordlund and Reichard, 2006). To avoid imbalanced levels of dNTPs and the increased mutation rates that are the inevitable consequences of this (Kumar et al, 2011; Mathews, 2006; Watt et al, 2016), RNRs are tightly controlled through transcriptional and Rozman Grinberg et al eLife 2018;7:e31529.
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