Biomimetic S‐Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes**

Abstract

S‐Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM‐dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S‐adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one‐pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin‐dependent radical SAM MT using S‐adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Enzymatic Synthesis of l‐Methionine Analogues and Application in a Methyltransferase Catalysed Alkylation Cascade**

“…In addition to the alternative starting materials discussed above, regeneration of the starting substrates from the byproduct SAH (including the starting substrate ATP) can reduce the costs, improve productivity and the atom economy. [8,[25][26][27] In addition to this extension of the substrate range, the scalability will be considered in future steps. So far, the reactions have not been performed on a preparative scale, which would be necessary for technical application.…”

Non-Native Site-Selective Enzyme Catalysis

“…While some radical SAM enzymes, including TsrM, which was actually found to employ a polar mechanism for methylation, have been explored as biocatalysts, such applications remain rare. To facilitate radical SAM biocatalysis by eliminating the need for stoichiometric SAM in these reactions, a SAM regeneration system described above (Scheme B) was further developed and used for selective alkylation of a glutamine residue in a 24-mer polypeptide ( 366 Scheme B) . This one-pot system involves ribophosphorylation of adenine and two subsequent phosphorylations to produce ATP, which is used by a methionine adenosyltransferase to produce SAM.…”

“…(A) General Scheme of the Native Prenyltransferase Reaction by Dimethylallyltryptophan Synthases (DMATSs) and Representative Non-Native Products for DMATSs with Different Site Selectivity; 375,383 residue in a 24-mer polypeptide (366 Scheme 57B). 391 This one-pot system involves ribophosphorylation of adenine and two subsequent phosphorylations to produce ATP, which is used by a methionine adenosyltransferase to produce SAM. This regeneration system was used with cobalamin-dependent glutamine C-methyltransferase (QCMT) to alkylate 366, showing the ability of this approach to generate SAM analogues by replacing the addition of methionine by ethionine (Scheme 57B).…”

Non-Native Site-Selective Enzyme Catalysis

“…To facilitate radical SAM biocatalysis by eliminating the need for stoichiometric SAM in these reactions, a SAM regeneration system described above (Scheme 12B) 72 was further developed and used for selective alkylation of a glutamine residue in a 24-mer polypeptide (366 Scheme 57B). 389 This one-pot system involves ribophosphorylation of adenine and two subsequent phosphorylations to produce ATP, which is used by a methionine adenosyltransferase to produce SAM. This regeneration system was used with cobalamindependent glutamine C-methyltransferase (QCMT) to alkylate 366, showing the ability of this approach to generate SAM analogues by replacing the addition of methionine by ethionine (Scheme 57B).…”

“…387 B) Alkylation of the glutamine residue in a 24-mer polypeptide substrate by QCMT employing a one-pot SAM regeneration system. 389 SAE: S-adenosylethionine, DOA: 5'deoxyadenosine, SAH: S-adenosylhomocysteine, Met: L-methionine, Eth: L-ethionine.…”