摘要：SummaryMethylation is envisioned as a promising way to rationally improve key pharmacokinetic characteristics of lead compounds. Although diverse tailoring enzymes are found to be clustered with cyclodipeptide synthases (CDPSs) to perform further modification reactions on the diketopiperazine (DKP) rings generating complex DKP-containing compounds, so far, a limited number of methyltransferases (MTs) co-occurring with CDPS have been experimentally characterized. Herein, we deciphered the methylation steps during drimentines (DMTs) biosynthesis with identification and characterization of DmtMT2-1 (fromStreptomycessp. NRRL F-5123) and DmtMT1 (fromStreptomyces youssoufiensisOUC6819). DmtMT2-1 catalyzesN4-methylation of both pre-DMTs and DMTs; conversely, DmtMT1 recognizes the DKP rings, functioning before the assembly of the terpene moiety. Notably, both MTs display broad substrate promiscuity. Their combinatorial expression with thedmt1genes in differentStreptomycesstrains successfully generated eight unnatural DMT analogs. Our results enriched the MT tool-box, setting the stage for exploring the structural diversity of DKP derivatives for drug development.Graphical AbstractDisplay OmittedHighlights•The methylation steps during drimentines biosynthesis were unraveled•TwoN-MTs with different regioselectivities were identified•The substrate promiscuities of DmtMT1 and DmtMT2-1 were probed•Combinatorial biosynthesis expanded the chemical space of drimentinesKinetic Chemistry; Enzyme Engineering; Structural Biology
