Abstract
Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) is a crucial human hydrolase that breaks down deoxynucleotides (dNTPs) and has important roles in HIV restriction, innate immunity, cancer, and cell cycle regulation. Activity of the human SAMHD1 (Hs SAMHD1) is tightly regulated and is contingent on tetramerization upon nucleotide binding. Recent crystal structures have challenged the widely accepted mechanism of a single Mg/Mn ion at the active site, revealing a metallocofactor containing two (if not three) metal ions, one of which is Fe. However, the role of Fe in catalysis and cofactor assembly is still unclear. SAMHD1 orthologs are present in various organisms, including viruses, plants, and pathogens, and play unknown roles in key biological processes. In this study, we focus on the SAMHD1 ortholog from Rhizophagus irregularis (Ri SAMHD1), a mycorrhizal fungus that promotes plant growth and nutrient uptake. Ri SAMHD1 assembles a redox-active diiron metallocofactor that hydrolyzes all four canonical dNTPs in the presence of excess Fe2+ or Mn2+. We also show that Ri SAMHD1 activity is not apparently dependent on a nucleotide-dependent oligomerization but is dependent on addition of exogenous metal ions suggesting a metal-driven regulation. Surprisingly, GTP and dGTP lead to activity inhibition at high concentrations. These findings provide insight into the molecular mechanisms of Ri SAMHD1 dNTP hydrolysis and highlight key differences to Hs SAMHD1, offering a first glimpse into the functional proteome of the SAMHD1-like landscape. Furthermore, because Ri SAMHD1 conserves the allosteric sites but not the requirement for oligomerization, it can serve as a simpler functional template for understanding Hs SAMHD1 regulation and activity.