Engineering Bifunctional Enzymes Capable of Adenylating and Selectively Methylating the Side Chain or Core of Amino Acids

Nonribosomal peptides (NRPs) are known sources of therapeutics. Some nonribosomal peptide synthetase assembly lines contain unique functional interrupted adenylation (A) domains, where nature has combined two different functional domains into one bifunctional enzyme. Most often these interrupted A domains contain a part of a methylation (M) domain embedded in their sequence. Herein, we aimed to emulate nature and create fully functional interrupted A domains by inserting two different noncognate M domains, KtzH(M_H) and TioS(M_3S), into a naturally occurring uninterrupted A domain, Ecm6(A₁T₁). We evaluated the engineered enzymes, Ecm6(A_1aM_HA_1bT₁) and Ecm6(A_1aM_3SA_1bT₁), by a series of radiometric assays and found that not only do they maintain A domain activity, but also they gain the site-specific methylation patterns observed in the parent M domain donors. These findings provide an exciting proof-of-concept for generating interrupted A domains as future tools to modify NRPs and increase the diversity and activity of potential therapeutics.