We present here a rapid, highly sensitive nonradioactive assay for adenylation enzyme selectivity determination and characterization. This method measures the isotopic back exchange of unlabeled pyrophosphate into γ-18O4-labeled ATP via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS), electrospray ionization liquid chromatography MS, or electrospray ionization liquid chromatography-tandem MS and is demonstrated for both nonribosomal (TycA, ValA) and ribosomal synthetases (TrpRS, LysRS) of known specificity. This low-volume (6 μl) method detects as little as 0.01% (600 fmol) exchange, comparable in sensitivity to previously reported radioactive assays and readily adaptable to kinetics measurements and high throughput analysis of a wide spectrum of synthetases. Finally, a previously uncharacterized A-T didomain from anthramycin biosynthesis in the thermophile S. refuinius was demonstrated to selectively activate 4-methyl-3-hydroxyanthranilic acid at 47°C, providing biochemical evidence for a new aromatic β-amino acid activating adenylation domain and the first functional analysis of the anthramycin biosynthetic gene cluster.
