Adenylation enzyme characterization using γ-18O4-ATP pyrophosphate exchange

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–O4-labeled ATP via MALDI-TOFMS, ESI-LC/MS or ESI-LC/MS/MS and is demonstrated for both nonribosomal (TycA, ValA) and ribosomal synthetases (TrpRS, LysRS) of known specificity. This low volume (6μL) 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.

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