Abstract2,5-Diketopiperazines are the smallest cyclic peptides comprising two amino acids connected via two peptide bonds. They can be biosynthesized in nature by two different enzyme families, either by nonribosomal peptide synthetases or by cyclodipeptide synthases. Due to the stable scaffold of the diketopiperazine ring, they can serve as precursors for further modifications by different tailoring enzymes, such as methyltransferases, prenyltransferases, oxidoreductases like cyclodipeptide oxidases, 2-oxoglutarate-dependent monooxygenases and cytochrome P450 enzymes, leading to the formation of intriguing secondary metabolites. Among them, cyclodipeptide synthase-associated P450s attracted recently significant attention, since they are able to catalyse a broader variety of astonishing reactions than just oxidation by insertion of an oxygen. The P450-catalysed reactions include hydroxylation at a tertiary carbon, aromatisation of the diketopiperazine ring, intramolecular and intermolecular carbon-carbon and carbon-nitrogen bond formation of cyclodipeptides and nucleobase transfer reactions. Elucidation of the crystal structures of three P450s as cyclodipeptide dimerases provides a structural basis for understanding the reaction mechanism and generating new enzymes by protein engineering. This review summarises recent publications on cyclodipeptide modifications by P450s.Key Points• Intriguing reactions catalysed by cyclodipeptide synthase-associated cytochrome P450s• Homo- and heterodimerisation of diketopiperazines• Coupling of guanine and hypoxanthine with diketopiperazines Graphical abstract

Heterologous expression of a silent gene cluster from Streptomyces cinnamoneus led to identification of guatrypmethine C, a guaninylated cyclo-L-Trp-L-Met derivative with two exo double bonds at the diketopiperazine ring. Structural elucidation was achieved using MS, 1 H, 13 C, and 15 N NMR analyses including 1 H− 13 C and 1 H− 15 N HMBC. Gene combination and biochemical investigation proved that the formation of the two CC bonds are catalyzed by two distinct enzyme families (i.e., cyclodipeptide oxidase and Fe II /2OG-dependent oxygenase).

Heterologous expression of a three-gene cluster from Streptomyces aurantiacus coding for a cyclodipeptide synthase, a prenyltransferase, and a methyltransferase led to the elucidation of the biosynthetic steps of streptoazine C (2). In vivo biotransformation experiments proved the high flexibility of the prenyltransferase SasB toward tryptophan-containing cyclodipeptides for regular C-3-prenylation. Furthermore, their corresponding dehydrogenated derivatives prepared by using cyclodipeptide oxidases were also used for prenylation. This study provides an enzyme with high substrate promiscuity from a less explored group of prenyltransferases for potential use to generate prenylated derivatives.

Tailoring enzymes are important modification biocatalysts in natural product biosynthesis. We report herein six orthologous two‐gene clusters for mycocyclosin and guatyromycine biosynthesis. Expression of the cyclodipeptide synthase genes gymA1–gymA6 in Escherichia coli resulted in the formation of cyclo‐l‐Tyr‐l‐Tyr as the major product. Reconstruction of the biosynthetic pathways in Streptomyces albus and biochemical investigation proved that the cytochrome P450 enzymes GymB1–GymB6 act as both intramolecular oxidases and intermolecular nucleobase transferases. They catalyze not only the oxidative C−C coupling within cyclo‐l‐Tyr‐l‐Tyr, leading to mycocyclosin, but also its connection with guanine and hypoxanthine, and are thus responsible for the formation of tyrosine‐containing guatyromycines, instead of the reported tryptophan‐nucleobase adducts. Phylogenetic data suggest the presence of at least 47 GymB orthologues, indicating the occurrence of a widely distributed enzyme class.

Cyclodipeptides from fungi and bacteria are often modified by different tailoring enzymes. They display various biological and pharmacological activities, and some derivatives are used as drugs. In a previous study, we elucidated the function of the silent guatrypmethine gene cluster from Streptomyces cinnamoneus containing a cyclodipeptide synthase (CDPS) core gene gtmA and four genes gtmB−gtmE for tailoring enzymes. The latter are used in this study for the design of modified cyclodipeptides by genetic engineering. Addition of six different cyclodipeptides to the Streptomyces albus transformant harboring gtmB−gtmE led to the detection of different pathway products. Coexpression of five CDPS genes from four Streptomyces strains with gtmB−gtmE resulted in the formation of diketopiperazine derivatives, differing in their modification stages. Our results demonstrate the potential of rational gene combination to increase structural diversity.