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A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst.

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Researchers engineered a key enzyme (thioesterase) in macrolide biosynthesis to overcome a catalytic bottleneck. This modification enables the production of novel epimerized macrolactone natural product analogs.

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Area of Science:

  • Biochemistry
  • Synthetic Biology
  • Enzyme Engineering

Background:

  • Macrolactonization of natural product analogs is challenging for biosynthesis and synthesis.
  • A thioesterase (TE) domain in the pikromycin (Pik) system was identified as a bottleneck for processing unnatural substrates, preventing epimerized macrolactone formation.

Purpose of the Study:

  • To understand the catalytic bottleneck in the Pik TE domain.
  • To engineer a stereoselective Pik TE variant with enhanced substrate flexibility and processing capabilities.
  • To enable the production of diastereomeric macrolactones.

Main Methods:

  • Molecular dynamics simulations to analyze substrate accommodation and conformation within the Pik TE active site.
  • Enzymatic engineering of the Pik TE by introducing the S148C mutation to create a gain-of-function variant (TES148C).
  • Quantum mechanical calculations to compare reaction mechanisms of wild-type (TEWT) and engineered TES148C.
  • Introduction of the S148C mutation into a polyketide synthase module (PikAIII-TE).

Main Results:

  • Molecular dynamics simulations revealed that while the epimerized hexaketide was accommodated, substrate conformations were predominantly unproductive, leading to hydrolysis.
  • The engineered TES148C variant showed improved reaction kinetics and gain-of-function processing of the unnatural, epimerized hexaketide.
  • Quantum mechanical analysis indicated a mechanistic shift in TES148C from stepwise addition-elimination to a lower-energy concerted acyl substitution compared to TEWT.
  • The mutated polyketide synthase module enabled the production of diastereomeric macrolactones.

Conclusions:

  • The engineered TES148C variant overcomes the catalytic bottleneck by altering the reaction mechanism, enabling efficient processing of unnatural substrates.
  • The study demonstrates a successful strategy for engineering enzyme specificity and flexibility in natural product biosynthesis.
  • This work provides a foundation for producing novel macrolactone analogs with potential therapeutic applications.