Biosynthesis and utilisation of SAM diastereomers as tools for the characterisation of SAM-dependent enzymes and product diversification

(S，S)-S-腺苷甲硫氨酸含有六个立体中心，其中四个通过腺苷部分的核糖构型固定。另一个立体中心是通过蛋氨酸的α-碳构型定义的，最后一个位于硫离子中心的立体中心是在SAM生物合成过程中由蛋氨酸腺苷转移酶(MAT)引入的。尤其是磺异构体，因为它是甲基转移酶(MTS)的抑制剂，而且是非酶作用的，特别是在体外应用中。α-碳异构体同样很有趣，因为它可能存在于用于产品多样化的蛋氨酸类似物的外消旋混合物中。此外，这两种类型的SAM非对映异构体都有可能在依赖SAM的酶催化的反应中产生不同的产物。本项目的重点是利用MATS、氯化酶和SAM水解酶生物催化合成相应的四种SAM非对映异构体，以及它们与不同类型的SAM依赖酶的相互作用，包括MTS、自由基SAM酶和使用SAM氨基羧丙基(ACP)的酶。在对计划中存在的不同类型的SAM依赖酶与SAM非对映异构体的接受性、底物结合和产物结果进行系统分析后，将探索SAM非对映异构体作为开发体内正交途径的策略的适用性。

(S,S)-S-adenosylmethionine (SAM) contains six stereocentres, four of those are fixed through the ribose configuration of the adenosine moiety. Another stereocentre is defined through the alpha-carbon configuration of methionine, and the last one at the sulfonium centre is introduced by methionine adenosyltransferase (MAT) during SAM biosynthesis. Especially the sulfonium epimer is of relevance, as it is an inhibitor for methyltransferases (MTs) and develops non-enzymatically, especially in in vitro applications. The alpha-carbon epimer is similarly interesting, as it might be present in racemic mixtures of methionine analogues used for product diversification. In addition, both types of SAM diastereomers have the potential to lead to different products in reactions catalysed by SAM-dependent enzymes. This project focuses on the biocatalytic synthesis of the corresponding four SAM diastereomers using MATs, chlorinases and SAM hydrolases, and their interaction with different types of SAM-dependent enzymes, covering MTs, radical SAM enzymes, and enzymes using the SAM aminocarboxypropyl (ACP) group. After a systematic analysis of different types of SAM-dependent enzymes present in the planned FOR with the SAM diastereomers regarding acceptance, substrate binding and product outcome, the suitability of SAM diastereomers as strategy for the development of orthogonal pathways in vivo will be explored.