Enzymatic generation of double-modified SAM analogues

甲硫氨酸腺苷转移酶（MAT）从甲硫氨酸和ATP产生S-腺苷-L-甲硫氨酸（SAM）。SAM作为大多数甲基转移酶（MT）的共底物，然而它们中的许多对于锍中心的甲基（或烷基或苄基）是混杂的。MT的广泛发生阻止了在锍中心具有扩展部分的SAM类似物用于研究单个MT或在细胞系统中选择性地标记生物分子。我们建议生成双修饰SAM类似物，其在锍中心和SAM的腺苷部分具有修饰。组合修饰应防止野生型MT的转化。我们的目标是通过工程化PC-MjMAT，一种来自詹氏甲烷球菌的MAT变体，对苄基甲硫氨酸类似物和许多ATP类似物具有高活性，以酶促方式产生这些双修饰的SAM类似物。具体而言，我们计划通过首先应用定点诱变然后定向进化来工程化PC-MjMAT变体以使用邻硝基苄基高半胱氨酸与GTP或2苯基乙炔基-ATP。新的MAT变体随后将在级联反应中与来自我们小组和其他FOR 5596合作伙伴的MT进行测试。我们将开始与定点突变，以提高两个MT（NovO和MTaq I）对双修饰SAM类似物在本授权期的最后阶段。最终，我们的目标是创建生物正交SAM类似物，以实现特定MT变体的转化，从而在细胞系统中进行选择性修饰。在细胞中选择性修饰靶分子的能力将为生物分子标记开辟一条新的途径，并且-与光可裂解基团（如邻硝基苄基部分）组合-甚至用于控制复杂细胞环境中的生物分子功能（例如表观遗传学）。

Methionine adenosyltransferases (MATs) produce S-adenosyl-L-methionine (SAM) from methionine and ATP. SAM serves as a cosubstrate for most methyltransferases (MTs), however many of them are promiscuous with regard to the methyl (or alkyl or benzyl) group at the sulfonium centre. The widespread occurrence of MTs prevents the use of SAM analogues with extended moieties at the sulfonium centre for studying individual MTs or labelling biomolecules selectively in cellular systems. We propose to generate double-modified SAM analogues with modifications at the sulfonium centre and the adenosine part of SAM. The combined modifications shall prevent the conversion by wildtype MTs. We aim to generate these double-modified SAM analogues enzymatically by engineering PC-MjMAT, a MAT variant from Methanocaldococcus jannaschii with high activity on benzylic methionine analogues and a number of ATP analogues. Specifically, we plan to engineer PC-MjMAT variants to use ortho-nitrobenzyl-homocysteine with GTP or 2 phenylethynyl-ATP by first applying site-directed mutagenesis and then directed evolution. The new MAT variants will subsequently be tested in cascade reactions with MTs from our group and other FOR 5596 partners. We will begin with site-directed mutations to enhance two MTs (NovO and MTaqI) toward the double-modified SAM analogues in the final stage of this grant period. Ultimately, we aim to create bioorthogonal SAM analogues to achieve conversion by a specific MT variant and thus selective modifications in cellular systems. The ability to selectively modify a target molecule in cells would open a new way for biomolecular labelling and - in combination with the photo-cleavable groups, such as the ortho-nitrobenzyl moiety - even for controlling biomolecular functions (e.g. in epigenetics) in the complex cellular environment.