Asparaginyl-tRNA synthetase pre-transfer editing assay

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Aminoacyl-tRNA synthetases (AARSs) are crucial enzymes for protein synthesis, ensuring accurate amino acid attachment to tRNA.
• AARSs possess editing mechanisms (pre-transfer and post-transfer) to prevent errors in protein synthesis.
• AARSs are validated targets for chemotherapy and natural antibiotics.

Purpose of the Study:

• To develop a high-throughput screening (HTS) assay for measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS).
• To identify inhibitors of AsnRS from natural product extracts for potential therapeutic applications.

Main Methods:

• Developed an HTS assay quantifying pyrophosphate formation via Malachite Green reaction to measure AsnRS pre-transfer editing.
• Utilized L-Aspartate-β-hydroxamate as an asparagine analogue to promote AsnRS editing.
• Screened 37,120 natural product extracts for inhibitors of Brugia malayi AsnRS (BmAsnRS).

Main Results:

• L-Aspartate-β-hydroxamate effectively promoted AsnRS editing activity (KM ~100 mM).
• Thiol agents (DTT, L-Cysteine) enhanced assay sensitivity by increasing BmAsnRS turnover.
• Identified several natural product extracts that inhibit BmAsnRS pre-transfer editing.

Conclusions:

• The developed HTS assay is effective for measuring AsnRS pre-transfer editing activity.
• The assay can be adapted for other enzymes with pre- or post-transfer editing functions.
• Identified potential lead compounds from natural products for developing new antimicrobial agents targeting AsnRS.