Enzymatic generation of double-modified SAM analogues

蛋氨酸腺苷转移酶(MAT)由蛋氨酸和三磷酸腺苷合成S-腺苷-L-蛋氨酸。SAM是大多数甲基转移酶(MTS)的共底物，但其中许多是关于硫中心的甲基(或烷基或苄基)的混杂。MTS的广泛出现阻碍了在细胞系统中使用具有扩展部分的SAM类似物来研究单个MTS或选择性地标记生物分子。我们建议通过在SAM的硫离子中心和腺苷部分进行修饰来产生双修饰的SAM类似物。组合修改应防止通过通配型MTS进行转换。我们的目标是通过工程PC-MjMAT来酶促产生这些双修饰的SAM类似物，PC-MjMAT是一种来自詹纳什甲烷球菌的MAT变体，对苄基蛋氨酸类似物和一些ATP类似物具有高活性。具体地说，我们计划通过首先应用定点突变，然后定向进化来设计PC-MjMAT变体，使其与GTP或2-苯乙炔-ATP一起使用邻硝基苄基-同型半胱氨酸。新的MAT变种随后将与我们小组和其他5596个合作伙伴的MTS进行级联反应测试。我们将从定点突变开始，以增强两个MT(Novo和MTaqI)，使其在此授权期的最后阶段成为双修饰的SAM类似物。最终，我们的目标是创建生物正交的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。选择性地修饰细胞中的目标分子的能力将为生物分子标记开辟一条新的途径，并与可光切割的基团相结合，例如邻硝基苄基，甚至用于在复杂的细胞环境中控制生物分子的功能(例如，在表观遗传学中)。

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.