Biochemical characterization and assay development for adenylosuccinate synthetase Rv0357c from Mycobacterium...

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• The rise of drug-resistant Mycobacterium tuberculosis (Mtb) necessitates novel antitubercular agents targeting essential pathways.
• The de novo purine biosynthesis pathway is critical for Mtb survival, making its enzymes, like adenylosuccinate (ADS) synthetase (ADSS), potential therapeutic targets.

Purpose of the Study:

• To biochemically characterize Mycobacterium tuberculosis adenylosuccinate (ADS) synthetase (ADSS) (Rv0357c).
• To develop robust, high-throughput screening (HTS)-compatible assays for Mtb ADSS activity to facilitate inhibitor discovery.

Main Methods:

• Expression and purification of recombinant Mtb ADSS using protein refolding and chaperone-assisted strategies.
• Enzymatic characterization using spectrophotometric, colorimetric, and fluorescence-based assays to determine kinetic parameters.
• Development and validation of nonradioactive MESG (5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) monophosphate assay) for monitoring GTP hydrolysis and phosphate release.

Main Results:

• Soluble, catalytically active Mtb ADSS was successfully obtained and characterized.
• Steady-state kinetic parameters were determined, confirming Michaelis-Menten kinetics.
• Scalable, nonradioactive assays were established and validated for HTS, successfully detecting inhibition by Aurodox.

Conclusions:

• This study provides the first comprehensive biochemical framework for Mtb ADSS.
• Developed assays are suitable for inhibitor screening and characterization.
• This work lays the foundation for structure-guided inhibitor discovery targeting purine biosynthesis for novel antitubercular strategies.