Extended Data Fig. 4: O-acyl-ADP-ribose can serve as an acyl donor.
From: Acylspermidines are conserved mitochondrial sirtuin-dependent metabolites
a, Scheme for comparing acyl transfer from acetyl-CoA and O-acetyl-ADPR to spermidine at neutral or slightly basic pH. Products of acyl transfer were derivatized with isobutyl chloroformate and analyzed by HPLC-MS. b, c, Estimation of the second-order rate constants for acyl transfer to form N1-acetylspermidine from AcCoA (b) or OAcADPR (c) at pH 8.0. Number of independent assays, n = 2. d, daspid#3 and daspid#4 are also produced from supplemented D4-glutaric acid. Representative of three experiments. e, Amounts of daspid#3 and daspid#4 produced from supplemented D4-glutaric acid (detected at m/z 306.2325) do not differ between WT and sir-2.3. Number of independent experiments, n = 3. f, Cartoon illustrating the production of N-glutarylspermidine from extracellular lysine and glutaric acid. g, Expression of human MacroD1 (ΔN58aa truncated) with an infusion thioredoxin and His6 tag in Rosetta(DE3) cells grown in LB induced by isopropyl β-D-1-thiogalactopyranoside. Elutions from a Ni-NTA column for recombinant protein purification are shown. h, TEV sequence was cleaved by TEV protease to release the MacroD1 for assays. Shown is the input and column-purified product. i, Steady-state kinetic analysis of OAcADPR and OGADPR hydrolysis catalyzed by human MacroD1 (ΔN58aa truncated) reveals both deacylation reactions follow saturation kinetics. The steady-state kinetic parameters Km and Vmax are determined by HPLC-MS for ADP-ribose formation to be 427 ± 74 µM and 0.17 ± 0.01 s−1 for OAcADPR, and 1020 ± 283 µM and 0.46 ± 0.08 s−1 for OGADPR. The reaction mixtures contain 0.5 µM MacroD1. Number of independent assays using the same batch of purified enzyme, n = 3. Data are mean ± s.d. in e and i. P values were calculated using unpaired, two-tailed t-tests with Welch correction in e. g and h are results from one experiment without attempts of replication.
