Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes

Abstract S ‐Adenosyl‐ l ‐methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM‐dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from “off‐the‐shelf” iodoalkanes through enzymatic alkylation of S ‐adenosyl‐ l ‐homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M −1 s −1 , convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S ‐alkylation of SAH with very high stereoselectivities (>99 % de ). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with “off‐the‐shelf” reagents.