Department of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
Angew Chem Int Ed Engl. 2020 Aug 24;59(35):14950-14956. doi: 10.1002/anie.202004963. Epub 2020 Jun 22.
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.
S-腺苷甲硫氨酸（SAM）依赖性甲基转移酶（MTs）催化各种小代谢物和生物大分子的甲基化。最近，发现了罕见的羧甲基化途径，包括利用由 SAM 通过 cxSAM 合酶（CmoA）生成的羧基-SAM（cxSAM）辅助因子的羧甲基转移酶酶。我们展示了 MT 酶如何利用 cxSAM 催化四氢异喹啉（THIQ）和儿茶酚底物的羧甲基化。通过定点突变创建了具有改进的 cxSAM 催化活性和选择性的正交 MT，随后与 CmoA 偶联导致更有效和选择性的羧甲基化。还开发了一种酶促方法来生成以前未描述的辅因子，即羧基-S-腺苷基-L-乙硫氨酸（cxSAE），从而能够将手性 1-羧乙基基团立体选择性地转移到底物上。
