Abstract
The pyrroloindole (hexahydropyrrolo[2,3-b]indole, HPI) structural motif is present in a wide range of natural products with various biological activities, yet its chemical synthesis poses a challenge, particularly regarding methylation at the indole C3 position. In nature, S-adenosyl methionine (SAM)-dependent methyltransferases efficiently catalyze this reaction with high stereoselectivity. This study presents the investigation and rational re-design of a potential methyltransferase, termed SeMT, from the actinomycete Saccharopolyspora erythraea. While its three-dimensional structure elucidated via X-ray crystallography confirmed extensive structural similarity to cyclic dipeptide-processing methyltransferases such as SgMT, its putative catalytic center is clearly divergent. Accordingly, wild-type SeMT displayed minimal activity with diketopiperazine (DKP) substrates, triggering an extensive mutagenesis effort aimed at iteratively enhancing this methyltransferase function. This work yielded a variant with appreciable activity, which was comprehensively characterized. Notably, a specific mutation within the catalytic triad of SeMT proved critical not only for its own function but also for the temperature-activity profile of its homolog protein SgMT. Beyond the specific properties of SeMT, these findings hence provide important insights into the active center architecture of indole C3-methyltransferases, supporting further development of these enzymes into refined biocatalysts for synthetic applications.