Complementary Fluorescent Probe Pair Targeting Histidinol Dehydrogenase Provides a Useful Tool for Target Validation

Area of Science:

• Agrochemical research
• Molecular biology
• Biochemistry

Background:

• Target-based pesticide design requires validation of novel candidate targets.
• Fluorescent probes are essential for studying molecular interactions and target functionality.

Purpose of the Study:

• To develop and utilize a fluorescent probe pair (HDP1 and HDP2) to assess the agrochemical potential of targeting histidinol dehydrogenase (HDH).
• To investigate the interactions between HDH, its natural substrate (HOL), and inhibitors.
• To visualize HDH activity and histidine biosynthesis pathway flux in vivo under various stress conditions.

Main Methods:

• Development of a complementary fluorescent probe pair, HDP1 and HDP2.
• Using HDP1 to probe inhibitor and substrate interactions with HDH.
• Employing HDP2 for in vivo imaging of HDH in Arabidopsis thaliana, Escherichia coli, and Saccharomyces cerevisiae.
• Analyzing changes in histidine biosynthesis pathway flux under stress and pesticide treatment.

Main Results:

• HDP1 demonstrated high sensitivity for detecting HDH interactions (0.17 microg/mL detection limit).
• HDP2 provided excellent in vivo imaging of HDH.
• HOL, the natural substrate, competitively binds to HDH, similar to HDH inhibitors.
• HDH inhibitors, other pesticides, and abiotic stresses impact histidine biosynthesis across different organisms.
• Stresses affect histidine biosynthesis via pentose phosphate pathway regulation and ATP-phosphoribosyltransferase expression inhibition.

Conclusions:

• Developing effective in vivo HDH inhibitors that compete with HOL remains a significant challenge.
• The histidine biosynthesis pathway's sensitivity to various stresses complicates HDH-targeted pesticide development.
• Environmental and cellular stresses can modulate the effectiveness of synthetic ligands targeting HDH.