The ARH and Macrodomain Families of α-ADP-ribose-acceptor Hydrolases Catalyze α-NAD⁺ Hydrolysis

Abstract

ADP-ribosyltransferases transfer ADP-ribose from β-NAD+ to acceptors; ADP-ribosylated acceptors are cleaved by ADP-ribosyl-acceptor hydrolases (ARHs) and proteins containing ADP-ribose-binding modules termed macrodomains. On the basis of the ADP-ribosyl-arginine hydrolase 1 (ARH1) stereospecific hydrolysis of α-ADP-ribosyl-arginine and the hypothesis that α-NAD+ is generated as a side product of β-NAD+/ NADH metabolism, we proposed that α-NAD+ was a substrate of ARHs and macrodomain proteins. Here, we report that ARH1, ARH3, and macrodomain proteins (i.e., MacroD1, MacroD2, C6orf130 (TARG1), Af1521, hydrolyzed α-NAD+ but not β-NAD+. ARH3 had the highest α-NADase specific activity. The ARH and macrodomain protein families, in stereospecific reactions, cleave ADP-ribose linkages to N- or O- containing functional groups; anomerization of α- to β-forms (e.g., α-ADP-ribosyl-arginine to β-ADP-ribose- (arginine) protein) may explain partial hydrolysis of ADP-ribosylated acceptors with an increase in content of ADP-ribosylated substrates. Af1521 and ARH3 crystal structures with bound ADP-ribose revealed similar ADP-ribose-binding pockets with the catalytic residues of the ARH and macrodomain protein families in the N-terminal helix and loop. Although the biological roles of the ARHs and macrodomain proteins differ, they share enzymatic and structural properties that may regulate metabolites such as α-NAD+.

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis

“…In 2017, MACROD1, MACROD2, and TARG1 were reported as being capable of removing ADP-ribose from both modified single-stranded DNA [63] and, in 2018, also being capable of removing ADP-ribose from MARylated single-stranded RNA [12]. Lastly, in 2019, the hydrolysis of α-NAD was added to the list of their in vitro activities [64].…”

NAD metabolism in aging and cancer

“…77–79 Both sets of enzymes cleave NAD at the N-glycosidic bond between nicotinamide and ADP-ribose, liberating free nicotinamide and covalently attaching the ADP-ribose moiety to protein amino acid side chains, including arginine, asparagine, glutamate, lysine, serine, and/or cysteine residues. 80,81 As discussed below, the poly(ADP-ribose) polymerases continue to cleave NAD and attach subsequent ADP-ribose moieties to growing chains of poly(ADP-ribose). 5…”

“…The mono(ADP-ribosyl)transferases (mARTs) are widely distributed among prokaryotes and eukaryotes where their function and regulatory selectivity appear to be related to their organ and subcellular distribution. 79,81 mARTs serve as a common mechanism for several prokaryotic toxins. 82 ADP-ribosylation of an arginine residue on a membrane-bound GTP-binding protein by cholera toxin or E. coli heat-labile enterotoxin increases the activity of adenylyl cyclase resulting in intestinal fluid and electrolyte flux leading to severe fluid loss and dehydration.…”

Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases

“…The assay utilizes a-NAD þ , which mimics protein ADP-ribosylation due to the anomerization of b-NAD þ . Indeed, several macrodomains have recently been shown to be able to hydrolyze a-NAD þ , 19 and despite that this does not measure removal of the ADP-ribosyl group from a protein, it provides a way to measure the hydrolysis activity of a substrate analogous to it. After enzymatic reaction, a-NAD þ is chemically converted to a fluorophore for quantification (Suppl.…”