pyrophosphate and 4-methyl-3-hydroxyanthranilic-acid

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 gamma-(18)O(4)-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 microl) 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 degrees C, providing biochemical evidence for a new aromatic beta-amino acid activating adenylation domain and the first functional analysis of the anthramycin biosynthetic gene cluster.

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Anthramycin; Biocatalysis; Chromatography, High Pressure Liquid; Diphosphates; Kinetics; Multigene Family; ortho-Aminobenzoates; Oxygen Radioisotopes; Peptide Synthases; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization