Substrate structure-activity relationships guide rational engineering of modular polyketide synthase ketoreductases

Area of Science:

• Biochemistry
• Organic Chemistry
• Enzymology

Background:

• Modular polyketide synthases (PKS) are crucial in natural product biosynthesis.
• Ketoreductases (KR) within PKS modules catalyze stereoselective reductions.
• Controlling stereochemistry in KR activity is vital for targeted synthesis.

Purpose of the Study:

• To investigate the structural basis of stereocontrol in modular PKS ketoreductases.
• To understand the role of specific enzyme-substrate interactions in dictating product stereochemistry.
• To engineer ketoreductase activity through targeted mutagenesis.

Main Methods:

• Performed a structure-activity relationship study using α-methyl, β-ketothioester substrates.
• Utilized four different ketoreductases, including the model enzyme EryKR1.
• Introduced mutations in residues identified as critical for contacting the β-ketoacyl moiety.

Main Results:

• Identified critical interactions between ketoreductases and the β-ketoacyl moiety of substrates.
• Demonstrated that specific mutations can significantly alter or reverse stereoselectivity.
• Two mutations in EryKR1 were sufficient to switch product formation from (2S,3R) to (2S,3S).

Conclusions:

• Enzyme-substrate interactions, particularly with the β-ketoacyl group, are paramount for stereocontrol in ketoreductases.
• Targeted mutagenesis of key residues offers a viable strategy for engineering ketoreductase stereoselectivity.
• This study provides insights into the mechanism of chiral center generation by modular PKS KRs.