Platform technologies for understanding and exploiting methylation.

This proposal seeks to develop a universal platform for the study and application of methionine adenosyltransferases (MATs) and methyltransferases (MTs) to facilitate MT-based biocatalysis/bioengineering of new chemical entities and provide tools relevant to the fundamental study of methylation in biology. The model MTs selected for this study represent broad catalytic diversity (C-methylation, O-methylation and N-methylation) and directly act upon a selected set of complex natural product-based drugs, validated clinical candidates or marketed agricultural products. The proposed studies will integrate the chemical synthesis and application of unique L-methionine (Met) analogs, MAT/MT structure determination, high throughput MAT/MT assay development/application and structure-guided MAT/MT directed evolution. The anticipated outcomes of this study include highly permissive/proficient MATs/MTs engineered for medicinal chemistry applications, new MAT/MT substrates/products, unprecedented differentially-alkylated analogs with potential therapeutic and/or agricultural applications, and an expanded understanding of MAT/MT structure-activity relationships of potential relevance to inhibitor design. Within this context, the proposed studies will also provide the first bioorthogonal strategy to exclusively interrogate or exploit a single MT within a cell containing a full complement of competing native MTs. This bioorthogonal platform will serve as a basis for engineering bacterial strains to produce differentially alkylated complex bioactive natural products, annotating function of bacterial biosynthetic MTs and set the stage for similar strategies to study methylation-dependent phenomena in the context of human disease or microbial/fungal/viral pathogenesis.