Adenylation enzyme characterization using gamma -(18)O(4)-ATP pyrophosphate exchange.
Chem Biol
; 16(5): 473-8, 2009 May 29.
Article
em En
| MEDLINE
| ID: mdl-19477411
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeo Sintases
/
Trifosfato de Adenosina
/
Difosfatos
/
Aminoacil-tRNA Sintetases
Idioma:
En
Revista:
Chem Biol
Assunto da revista:
BIOLOGIA
/
BIOQUIMICA
/
QUIMICA
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos