Jan 10, 2021

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

Posted by in categories: biotech/medical, engineering

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 Å 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”).

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