Engineering and Directed Evolution of DNA Methyltransferases

Abstract

DNA methyltransferases (MTases) constitute an attractive target for protein engineering, thus opening the road to new ways of manipulating DNA in a unique and selective manner. Here, we review various aspects of MTase engineering, both methodological and conceptual, and also discuss future directions and challenges. Bacterial MTases that are part of restriction/modification (R/M) systems offer a convenient way for the selection of large gene libraries, both in vivo and in vitro. We review these selection methods, their strengths and weaknesses, and also the prospects for new selection approaches that will enable the directed evolution of mammalian DNA methyltransferases (Dnmts). We explore various properties of MTases that may be subject to engineering. These include engineering for higher stability and soluble expression (MTases, including bacterial ones, are prone to misfolding), engineering of the DNA target specificity, and engineering for the usage of S-adenosyl-L-methionine (AdoMet) analogs. Directed evolution of bacterial MTases also offers insights into how these enzymes readily evolve in nature, thus yielding MTases with a huge spectrum of DNA target specificities. Engineering for alternative cofactors, on the other hand, enables modification of DNA with various groups other than methyl and thus can be employed to map and redirect DNA epigenetic modifications.

Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites

“…This method, originally devised for cloning DNA MTase genes ( 44 ) and complete restriction-modification systems ( 45 ), was later adopted for the selection of mutant MTases ( 46 , 47 ) notably those with altered specificity ( 8 , 37–39 , 41 , 48 ). Great advantages of the method are the physical linkage between the mutant genes and their encoded phenotypes manifested in the methylation patterns of the plasmid, and the powerful selection provided by restriction digestion ( 3 , 49 ).…”

Revealing Drivers for Carboxy-S-adenosyl-l-methionine Use by Neomorphic Variants of a DNA Methyltransferase

“…While the present study is rooted in a rational-design approach with molecular dynamics simulations guiding selection of mutagenesis candidates, our approach could be complemented by random mutagenesis studies or direct coupling analysis, which might further enhance the selectivity and/or activity profiles of CxMTases similar to those that have been used for other MTases. − Directed evolution in particular has become one of the most powerful and widespread tools for improving desired protein function, especially when a selective pressure is applied . Indeed, the fact that the CxMTase activity can be observed in E. coli makes it viable that selection strategies could be employed to discover mutants that further alter either enzymatic efficiency or selectivity in favor of CxSAM over SAM.…”

Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants

“…Crystal structures and computational studies have provided us with knowledge about the reaction mechanisms and have identified critical active-site residues that influence the functional properties. This knowledge has enabled many research groups to use protein engineering methods to tailor active sites of enzymes for altered efficiency, substrate specificity or other desired features ( Norrgård et al , 2006 ; Laurino et al , 2016 ). However, currently used methods are limited in efficacy due to lack of solved protein structures and incomplete knowledge of the intricacies of enzyme catalysis.…”