Rational Engineering of Phospholipase D Unlocks Robust Catalysis for Phosphatidylserine Formation

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Phosphatidylserine (PS) is crucial for brain health and cognitive function, potentially mitigating Alzheimer's disease.
• Enzymatic synthesis of PS faces challenges in suppressing hydrolysis and enhancing phospholipase D (PLD)-catalyzed transphosphatidylation.
• Developing efficient and stable enzymes is key for green and cost-effective PS production.

Purpose of the Study:

• To engineer Streptomyces antibioticus phospholipase D (SaPLD) for enhanced phosphatidylserine (PS) yield and stability.
• To elucidate the structure-mechanism-function relationships governing SaPLD activity.
• To establish a robust enzymatic process for high-efficiency PS production.

Main Methods:

• Mechanism-guided protein engineering of SaPLD based on structural insights.
• Site-directed mutagenesis (W187I, V380W, G381A) to create SaPLD variants.
• Enzyme characterization, including yield determination (up to 95.8% PS), thermostability, and kinetic analysis.
• Large-scale fermentation (5 L) for enzyme production.
• Molecular dynamics (MD) simulations and solvent contact/energy analyses.

Main Results:

• The W187I mutation significantly increased PS yield to 58.3%.
• The V380W mutation enhanced enzyme thermostability.
• The combined mutant SaPLD-R7 (W187I/V380W/G381A) achieved a maximum PS yield of 95.8%, overcoming the activity-stability trade-off.
• MD simulations revealed that SaPLD-R7 exhibits improved substrate binding and catalysis due to shortened active site distances and reduced local flexibility.
• Enhanced stability was confirmed through solvent contact and energy analyses.

Conclusions:

• A structure-mechanism-function framework was established for enhancing PLD-catalyzed transphosphatidylation.
• Engineered SaPLD-R7 provides a highly efficient and stable enzymatic route for phosphatidylserine production.
• This study offers a valuable method for producing functional food ingredients like PS.