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Asparaginyl-tRNA synthetase pre-transfer editing assay. Curr Drug Discov Technol 2011 Mar;8(1):66-75

Date

11/26/2010

Pubmed ID

21091430

Abstract

Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-β-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 mM. The assay sensitivity was enhanced by the thiol agents, DTT and L-Cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.

Author List

Danel F, Caspers P, Nuoffer C, Härtlein M, Kron MA, Page MG

Author

Michael Kron MD Director, Professor in the Medicine department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acids
Amino Acyl-tRNA Synthetases
Animals
Asparagine
Aspartate-tRNA Ligase
Brugia malayi
Coloring Agents
Cysteine
Drug Discovery
High-Throughput Screening Assays
Plant Extracts
RNA Editing
RNA, Transfer
RNA, Transfer, Amino Acyl
Rosaniline Dyes
Staphylococcus epidermidis
jenkins-FCD Prod-388 89e904233d719332173309c68ab82b0b2a78a3a7