PDF(Pubmed)
Abstract：
Methylation, a widely occurring natural modification serving diverse regulatory and structural functions, is carried out by a myriad of S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). The AdoMet cofactor is produced from l-methionine (Met) and ATP by a family of multimeric methionine adenosyltransferases (MAT). To advance mechanistic and functional studies, strategies for repurposing the MAT and MTase reactions to accept extended versions of the transferable group from the corresponding precursors have been exploited. Here, we used structure-guided engineering of mouse MAT2A to enable biocatalytic production of an extended AdoMet analogue, Ado-6-azide, from a synthetic methionine analogue, S-(6-azidohex-2-ynyl)-l-homocysteine (N3-Met). Three engineered MAT2A variants showed catalytic proficiency with the extended analogues and supported DNA derivatization in cascade reactions with M.TaqI and an engineered variant of mouse DNMT1 both in the absence and presence of competing Met. We then installed two of the engineered variants as MAT2A-DNMT1 cascades in mouse embryonic stem cells by using CRISPR-Cas genome editing. The resulting cell lines maintained normal viability and DNA methylation levels and showed Dnmt1-dependent DNA modification with extended azide tags upon exposure to N3-Met in the presence of physiological levels of Met. This for the first time demonstrates a genetically stable system for biosynthetic production of an extended AdoMet analogue, which enables mild metabolic labeling of a DNMT-specific methylome in live mammalian cells.
摘要：
甲基化,一种广泛发生的自然修饰，服务于不同的监管和结构功能，由无数的S-腺苷-1-甲硫氨酸(AdoMet)依赖性甲基转移酶(MTases)进行。AdoMet辅因子由1-甲硫氨酸(Met)和ATP通过多聚体甲硫氨酸腺苷转移酶(MAT)家族产生。为了推进机械和功能研究，已经开发了重新利用MAT和MTase反应以接受来自相应前体的可转移基团的扩展版本的策略。这里,我们使用小鼠MAT2A的结构引导工程来实现生物催化生产扩展的AdoMet类似物，Ado-6-叠氮化物,从合成的蛋氨酸类似物，S-(6-叠氮基己-2-炔基)-1-高半胱氨酸(N3-Met)。在不存在和存在竞争性Met的情况下，三种工程改造的MAT2A变体在与M.TaqI和小鼠DNMT1的工程改造变体的级联反应中显示出催化能力，并支持DNA衍生化。然后，我们通过使用CRISPR-Cas基因组编辑将两种工程化变体作为MAT2A-DNMT1级联安装在小鼠胚胎干细胞中。所得细胞系维持正常的生存力和DNA甲基化水平,并且在生理水平的Met存在下暴露于N3-Met时显示出Dnmt1依赖性DNA修饰,具有延伸的叠氮化物标签。这首次证明了用于生物合成生产扩展AdoMet类似物的遗传稳定系统，这使得在活哺乳动物细胞中DNMT特异性甲基化体的温和代谢标记成为可能。
