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Enzymatic Generation of Double‐Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases

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An engineered MAT (methionine adenosyltransferase) is capable of generating double-modified AdoMet analogues (DM-AdoMets) when provided with non-natural methionine analogues and nucleoside-modified ATP analogues. DM-AdoMets have unique properties and not all MTases (methyltransferases) can easily convert them. This increases selectivity for MTase-based biomolecular labelling applications, enabling differential MTase targeting wherein multiple MTases are present (i. e., in cell).


Abstract

Methyltransferases (MTases) have become an important tool for site-specific alkylation and biomolecular labelling. In biocatalytic cascades with methionine adenosyltransferases (MATs), transfer of functional moieties has been realized starting from methionine analogues and ATP. However, the widespread use of S-adenosyl-l-methionine (AdoMet) and the abundance of MTases accepting sulfonium centre modifications limit selective modification in mixtures. AdoMet analogues with additional modifications at the nucleoside moiety bear potential for acceptance by specific MTases. Here, we explored the generation of double-modified AdoMets by an engineered Methanocaldococcus jannaschii MAT (PC-MjMAT), using 19 ATP analogues in combination with two methionine analogues. This substrate screening was extended to cascade reactions and to MTase competition assays. Our results show that MTase targeting selectivity can be improved by using bulky substituents at the N6 of adenine. The facile access to >10 new AdoMet analogues provides the groundwork for developing MAT-MTase cascades for orthogonal biomolecular labelling.

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