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Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications.

Yonghui Xie1, Changyu Zhang2, Zhihua Wang3

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A novel fluorogenic assay for acetohydroxyacid synthase (AHAS) enables high-throughput screening of inhibitors. This assay aids in developing herbicides, identifying herbicide-resistant genes, and discovering potential antiobesity therapeutics targeting gut bacteria.

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Area of Science:

  • Biochemistry
  • Enzyme Assays
  • Drug Discovery

Background:

  • Acetohydroxyacid synthase (AHAS) is a crucial enzyme in plants and microorganisms, absent in mammals, making it a prime drug target.
  • Developing efficient, high-throughput screening methods for AHAS inhibitors is essential for drug discovery and agricultural applications.

Purpose of the Study:

  • To develop the first fluorogenic assay for AHAS utilizing an intramolecular charge transfer (ICT) fluorescent probe.
  • To demonstrate the assay's utility in high-throughput screening for herbicides, identifying herbicide resistance, and discovering selective inhibitors of microbial AHAS.

Main Methods:

  • Development of a novel ICT-based fluorescent probe for AHAS activity detection.
  • Application of the fluorogenic assay for screening commercial herbicides against various AHAS enzymes and mutants.
  • Utilizing the assay to identify herbicide resistance in soybean AHAS and selective inhibitors for intestinal bacteria.

Main Results:

  • The developed fluorogenic assay is facile, sensitive, continuous, and applicable to diverse AHAS sources, including crude cell lysates.
  • Successful high-throughput screening identified herbicide-matching strategies and a soybean AHAS gene conferring broad-spectrum resistance.
  • Selective inhibitors targeting intestinal bacterial AHAS were identified, including an agent inhibiting obesity-associated Ruminococcus torques.

Conclusions:

  • The novel fluorogenic AHAS assay provides a powerful tool for high-throughput screening in various AHAS-related research areas.
  • The findings suggest potential applications of AHAS inhibitors as antiobesity therapeutics by targeting specific gut microbes.
  • The assay's design principle may be extendable to developing fluorogenic assays for other enzyme classes.