Design of highly active substrates using molecular docking for microbial transglutaminase detection

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Transglutaminase (TGase) enzymes catalyze crucial protein cross-linking and modification reactions.
• Developing highly active TGase substrates is essential for various biotechnological applications.
• Microbial transglutaminase (mTGase) serves as a model enzyme for studying TGase family interactions.

Purpose of the Study:

• To design and identify novel, high-activity peptide substrates for microbial transglutaminase (mTGase).
• To leverage enzyme-substrate interaction principles combined with computational and experimental approaches.

Main Methods:

• Utilized molecular docking simulations to predict potential substrate interactions with mTGase.
• Synthesized and experimentally screened twenty-four peptide substrates for catalytic activity.
• Assessed substrate performance under physiological conditions (37 °C, pH 7.4).

Main Results:

• All twenty-four designed peptide substrates exhibited good catalytic activity with mTGase.
• The peptide sequence FFKKAYAV (acyl acceptor) and VLQRAY (acyl donor) showed optimal reaction efficiency.
• This optimized substrate combination enabled highly sensitive detection of mTGase down to 26 nM.
• Substrate groupings KAYAV and AFQSAY detected 130 nM mTGase, showing 20-fold higher activity than natural collagen substrate.

Conclusions:

• A combined approach of molecular docking and traditional experiments is effective for designing high-activity TGase substrates.
• The developed peptide substrates demonstrate superior performance and sensitivity compared to natural substrates.
• These findings highlight the potential for creating tailored substrates for specific TGase applications under physiological conditions.