Engineered Methionine Adenosyltransferase Cascades for Metabolic Labeling of Individual DNA Methylomes in Live Cells.

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.