Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways

Abstract

S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy‐SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site‐directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co‐factor, carboxy‐S‐adenosyl‐l‐ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1‐carboxyethyl group to the substrate.

A bicyclic S-adenosylmethionine regeneration system applicable with different nucleosides or nucleotides as cofactor building blocks

“…Furthermore, the regeneration system is a starting point for application in bioorthogonal systems. 5,19,24,26,29 Especially when using lysates, it will be possible to optimise parameters such as the enzyme/crude lysate loading and the ratio of substrate to cofactor building block for a specific enzyme-substrate combination in order to achieve the highest conversion possible with an adequate TTN. In combination with an efficient purification strategy, selectively methylated products could also be sustainably produced on a large biocatalytic scale.…”

“…The biocatalytic application of MTs, also using SAM analogues, [8][9][10][11][12] are promising starting points for sustainable methods. 13,14 While SAM analogues allowing to transfer larger alkyl chains have been widely employed for product diversification, [15][16][17][18][19][20][21][22][23][24][25] the variation of the nucleobase has become a new focus, especially regarding the long-term goal to make bioorthogonal systems available. [25][26][27][28][29] The limitations of using SAM are its instability under physiological conditions, i.e.…”

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs

“…SAM analogs enabling these diverse alkylation reactions are crucial not only for expanding the industrial relevance of biocatalytic alkylation but also for discovering promiscuous MTs [1c, 6] . However, very few naturally occurring SAM analogs [5a, 6, 7] are known, making limited access to SAM analogs one of the most serious impediments to progress in the field. With the exception of S ‐adenosyl‐ l ‐ethionine (SAE), SAM analogs are not readily available from commercial suppliers and have to be prepared on demand.…”

Engineered Enzymes Enable Selective N‐Alkylation of Pyrazoles With Simple Haloalkanes

“…Recently,s ignificant progress towards enzymatic alkylation chemistry using "off the shelf" alkylation reagents has been made,inparticular exploiting SAM-dependent methyltransferases (MTs). MTs use the cosubstrates S-adenosyl-lmethionine (SAM) and an atural carboxy-analog [17] of SAM in highly selective methylation and carboxymethylation reactions.M ultiple studies have further demonstrated that MTs are functional with non-natural SAM analogs (NSA) to selectively transfer ah uge variety of alkyl groups. [11,18] However,t he synthesis of NSA is currently not particularly straight forward ( Figure S4).…”