Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 angstrom revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification (writing) of plasmid DNA and light-triggered removal (erasing).

Automated summary

Exploring the methylation and demethylation of DNA, RNA, and proteins as a major regulatory mechanism benefits from the generation of AdoMet analogs with photocaging groups, allowing for site-specific inhibition and timed removal of methyltransferases. The engineering of SAM synthetase from Cryptosporidium hominis enables the efficient generation of non-aliphatic AdoMet analogs, which can be used for sequence-specific modification and light-triggered removal of DNA and RNA modifications.