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

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.

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

“…The chemical and enzymatic synthesis methods of FAD nucleobase analogues we report here form the basis of production of artificial fluorescent and other non‐natural FAD variants in the future. These FAD analogues, similar to SAM and NAD + analogues reported in literature, may have interesting applications in drug development, cell imaging, and biotechnology applications such as bioremediation [43–51] . For example, a diverse library of SAM analogues was created and tested as cofactors in methyltransferase enzymes implicated in small molecule and RNA methylations [51] .…”

“…These FAD analogues, similar to SAM and NAD + analogues reported in literature, may have interesting applications in drug development, cell imaging, and biotechnology applications such as bioremediation. [43][44][45][46][47][48][49][50][51] For example, a diverse library of SAM analogues was created and tested as cofactors in methyltransferase enzymes implicated in small molecule and RNA methylations. [51] In another instance, cytosine analogues of NAD + have been used as biorthogonal redox systems with engineered enzymes formate dehydrogenase and malic enzyme for CO 2 fixation in E. coli.…”

“…[43][44][45][46][47][48][49][50][51] For example, a diverse library of SAM analogues was created and tested as cofactors in methyltransferase enzymes implicated in small molecule and RNA methylations. [51] In another instance, cytosine analogues of NAD + have been used as biorthogonal redox systems with engineered enzymes formate dehydrogenase and malic enzyme for CO 2 fixation in E. coli. [28,32,33] Likewise, we envision the use of FAD nucleobase analogues as biorthogonal catalysts for the study of specific enzyme mechanisms in vivo and as biochemical probes to investigate cellular metabolism.…”

Post-synthetic benzylation of the mRNA 5′ cap via enzymatic cascade reactions

“…1C), and solved the crystal structures in complex with an ortho -nitrobenzyl (ONB)-containing AdoMet analogue (AdoONB) and derivatives. 14,20…”

Biocatalytic Ether Lipid Synthesis by an Archaeal Glycerolprenylase

“…As such, there is great interest in expanding the toolbox of ether synthases, which can operate under mild, aqueous conditions and with largely unmet regioselectivity. Consequently, tremendous progress has recently been made with biocatalytic alkylations using engineered methyltransferases and SAM-analogues [8][9][10][11][12][13][14] as well as engineered peptide prenyltransferases. 7,[15][16][17] In contrast to the wealth of work on these enzyme families, other naturally occurring ether synthases are comparably sparsely researched.…”