Mechanism of the metal and substrate selectivity of human ADP-ribosyl-acceptor hydrolase 3 (ARH3)

ARH3 is a Mg²⁺-containing multifunctional metalloenzyme that hydrolyzes various ADP-ribosylated substrates including poly(ADP-ribose) (PAR), serine mono-ADP-ribosylation, and α-NAD⁺. ARH3^–/– cells show an increased cellular ADP-ribosylation and concomitant increased cell death upon DNA damages, suggesting ARH3 functions as the key determinant for cell fate and ARH3 can be an attractive therapeutic target for cancers with elevated ADP-ribosylations. However, the basis of metal and substrate selectivity and the role of each Mg²⁺ (Mg_A and Mg_B) remain poorly understood. Here, using structural, mutational, and quantitative biochemical tools, including our newly developed NUDT5-coupled AMP/Glo assay that selectively monitors the protein-free ADP-ribose, we show that flexible ARH3 undergoes conformational changes upon substrate-binding and two Mg²⁺ ions show distinctive roles during catalysis. While Mg_B is important for specific substrate binding, Mg_A is key to precisely aligning substrates for catalysis. Our new ARH3-substrate complex structure reveal two different modes of action and mutational analysis supports this model. Our collective results highlight unique roles of two Mg²⁺ ions in ARH3, propose key catalytic residues, and lay the foundation to develop specific ARH3 inhibitors.

Figure 1. Substrate and structure of human ARH3.

Figure 2. Selective monitoring of protein-free ADP-ribose.