A tRNA-Dependent Two-Enzyme Pathway for the Generation of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines

A large number of bioactive natural products containing a 2,5-diketopiperazine (DKP) moiety have been isolated from various microbial sources. Especially tryptophan-containing cyclic dipeptides (CDPs) show great structural and functional diversity, while little is known about their biosynthetic pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide synthase (CDPS)-containing gene cluster from Actinosynnema mirum spanning 2.9 kb that contains two putative DKP-modifying enzymes. We establish the biosynthetic pathway leading to two methylated ditryptophan CDPs through in vivo and in vitro analyses. Our studies identify the first CDPS (Amir_4627) that shows high substrate specificity synthesizing only one main product, cyclo(Trp-Trp) (cWW). It is the first member of the CDPS family that can form ditryptophan DKPs and the first prokaryotic CDPS whose main product constituents differ from the four amino acids (Phe, Leu, Tyr, and Met) usually found in CDPS-dependent CDPs. We show that after cWW formation a S-adenosyl-l-methionine-dependent N-methyltransferase (Amir_4628) conducts two successive methylations at the DKP-ring nitrogens and additionally show that it is able to methylate four other phenylalanine-containing CDPs. This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase. The large number of known modifying enzymes of bacterial and fungal origin known to act upon Trp-containing DKPs makes the identification of a potent catalyst for cWW formation, encoded by a small gene, valuable for combinatorial in vivo as well as chemoenzymatic approaches, with the aim of generating derivatives of known CDP natural products or entirely new chemical entities with potentially improved or new biological activities.