Efficient transferase engineering for SAM analog synthesis using combinatorial library design and high-throughput...

Area of Science:

• Biocatalysis and enzyme engineering
• Organic synthesis
• Medicinal chemistry

Background:

• Alkylation reactions are crucial in chemical synthesis and pharmaceuticals but often lack selectivity.
• Conventional methods struggle with chemo- and regioselectivity, limiting their application.
• Biocatalytic alkylation using S-adenosyl-ʟ-methionine (SAM)-dependent methyltransferases (MTs) offers a highly selective alternative.

Purpose of the Study:

• To develop an efficient engineering approach for creating highly active methyltransferase (MT) variants.
• To expand the substrate scope of MTs for synthesizing diverse S-adenosyl-ʟ-methionine (SAM) analogs using various haloalkanes.
• To overcome limitations in current biocatalytic alkylation strategies.

Main Methods:

• Designed a combinatorial mutant library targeting multiple active site residues in parallel using sequence information and protein modeling.
• Generated the mutant library using synthetic DNA fragments, leveraging natural sequence diversity.
• Employed high-throughput liquid chromatography-mass spectrometry (LC/MS) for screening and identifying active enzyme variants.

Main Results:

• Identified enzyme variants with orders-of-magnitude higher activity compared to the wild-type enzyme.
• Successfully demonstrated the synthesis of several SAM analogs from diverse haloalkanes.
• Achieved significant improvements in enzyme activity and substrate scope for biocatalytic alkylation.

Conclusions:

• The presented engineering approach is effective for enhancing methyltransferase (MT) activity and broadening substrate specificity.
• This method holds promise for the development of novel biocatalysts for synthesizing SAM analogs and other valuable compounds.
• The strategy can be applied to engineer other enzyme classes beyond methyltransferases.