A tRNA-Dependent Two-Enzyme Pathway for the Generation
of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines
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Abstract
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 <i>Actinosynnema
mirum</i> spanning 2.9 kb that contains two putative DKP-modifying
enzymes. We establish the biosynthetic pathway leading to two methylated
ditryptophan CDPs through <i>in vivo</i> and <i>in
vitro</i> 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 <i>S</i>-adenosyl-l-methionine-dependent <i>N</i>-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 <i>in vivo</i> 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