Adenylation enzyme characterization using gamma -(18)O(4)-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 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.
Phelan
Vanessa V
VV
Department of Chemistry, Vanderbilt University, Nashville, TN 37204, USA.
Du
Yu
Y
McLean
John A
JA
Bachmann
Brian O
BO
D000255:Adenosine Triphosphate / Q000138:chemical synthesis / Q000737:chemistry*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D000876:Anthramycin / Q000096:biosynthesis / Q000737:chemistry; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D011756:Diphosphates / Q000737:chemistry*; D007700:Kinetics; D005810:Multigene Family; D010104:Oxygen Radioisotopes; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D062367:ortho-Aminobenzoates / Q000378:metabolism
D011756:Diphosphates; D010104:Oxygen Radioisotopes; D062367:ortho-Aminobenzoates; D000876:Anthramycin; C034508:4-methyl-3-hydroxyanthranilic acid; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase