Characterisation and engineering of the cobalamin-dependent Radical SAM methyltransferase Orf29 for the synthesis of novel SAM derivatives

不寻常的，含环丙烷的氨基酸（1 S，2S）-2-甲基-1-氨基环丙烷-1-羧酸（（1 S，2S）-MeACC）是在两步酶促过程中生物合成的，该过程使用S-腺苷-L-甲硫氨酸（SAM）作为初始底物。在第一步中，钴胺素依赖性自由基SAM甲基转移酶（B12-RSMT），称为Orf 29，催化SAM的甲硫氨酸部分的甲基化，产生（4“R）-4”-甲基-SAM。在第二步中，MeACC合酶Orf 30负责环化反应，导致形成（1 S，2S）-MeACC。在这个项目中，B12-RSMT Orf 29将被表征，以阐明这种有趣的酶的结构-功能关系，它利用SAM在三个不同的角色：作为辅因子引发自由基催化，作为甲基基团供体和作为底物。为了理解Orf 29的催化机制，我们的目标是阐明各个反应步骤的顺序，并定义SAM在活性位点的结合位点，这使得结合的SAM履行其独特的作用。除了Orf 29的功能和结构表征之外，我们的目标是使用这种酶作为平台来合成不同的SAM衍生物。为此，将使用生物化学、结构和生物信息学方法。最后，Orf 29变体可在未来用于SAM衍生物的生物正交合成，以掺入到新型的修饰的肽抗生素中。

The unusual, cyclopropane-containing amino acid (1S,2S)-2-methyl-1-aminocyclopropane-1-carboxylic acid ((1S,2S)-MeACC) is biosynthesised in a two-step enzymatic process that utilises S-adenosyl-L-methionine (SAM) as initial substrate. In the first step, a cobalamin-dependent Radical SAM methyltransferase (B12-RSMT), termed Orf29, catalyses the methylation of the methionine moiety of SAM yielding (4’’R)-4’’-methyl-SAM. In the second step, the MeACC synthase Orf30 is responsible for the cyclisation reaction leading to formation of (1S,2S)-MeACC. In this project, the B12-RSMT Orf29 will be characterized in order to elucidate the structure-function relationship of this intriguing enzyme, which utilizes SAM in three distinct roles: as cofactor for initiation of radical catalysis, as methyl group donor and as substrate. In order to understand the catalytic mechanism of Orf29, we aim to elucidate the sequence of individual reaction steps and define the binding sites for SAM in the active site, which allow the bound SAM to fulfil its distinct roles. Besides the functional and structural characterization of Orf29, we aim to use this enzyme as a platform to synthesize diverse SAM derivatives. For this purpose, biochemical, structural and bioinformatic approaches will be used. Finally, Orf29 variants may be used in the future for the bioorthogonal synthesis of SAM derivatives for incorporation into novel, modified peptide antibiotics.