Comparative studies on similarities and differences of cyclodipeptide oxidases for installation of C–C double bonds at the diketopiperazine ring

Abstract

Cyclodipeptide oxidases (CDOs) perform dehydrogenations on diketopiperazines and play an important role in the cyclodipeptide diversification. In this study, we investigated the two known CDOs AlbA/B and Ndas_1146/7 and one new member, CDO-Np. LC-MS monitoring of 32 cyclodipeptide biotransformations in E. coli revealed good consumption of cyclodipeptides containing aromatic amino acids. Cyclodipeptides consisting solely of aliphatic amino acids were poor substrates. In vitro assays of 34 substrates with crude enzyme extracts and product identification proved that the CDO-Np-containing extract catalyzes the formation of two CC double bonds in many cases. The extracts containing the two other enzymes had lower activities and catalyzed mainly didehydrogenations. For didehydrogenation, the phenylalanyl or tyrosyl site was usually preferred. No or very low acceptance of benzodiazepinediones and a 2,6-diketopiperazine proved the importance of the 2,5diketopiperazine ring. N-Methylation at the diketopiperazine ring or prenylation of the tryptophan-containing cyclodipeptides influences the enzyme activity and product spectrum. Key points • Comparison of catalytic activities of three enzymes; Diverse cyclodipeptides and derivatives as substrates; Determination of double bond formation using 2 H-labeled substrates; Product identification also by interpretation of MS 2 fragmentation pattern.

In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

“…The same was true for cLL, cFL, and cYY. Finally, the number of dehydrogenated derivatives detected for a given m/z value ranged from one to three, as previously observed for characterized CDOs [14,18,30], and were initially differentiated by their retention time. One interesting point concerns the utility of these CDOs to produce a desired derivative in large quantities.…”

“…Recently, S. M. Li et al assessed the activity of three CDOs, AlbA/B, Ndas_1146/1147, and CDO-Np from Nocardiopsis prasina (CDO- N spL17 in Fig. 2 and Additional file 1 : Table S1) on chemically synthesized CDPs in either in vitro assays using CDO-containing cell-free extracts or in feeding experiments to E. coli bacteria expressing each CDO [ 14 ]. The authors mentioned the difficulty of the three CDOs to generate ∆Trp-containing derivatives [ 14 ].…”

“…2 and Additional file 1 : Table S1) on chemically synthesized CDPs in either in vitro assays using CDO-containing cell-free extracts or in feeding experiments to E. coli bacteria expressing each CDO [ 14 ]. The authors mentioned the difficulty of the three CDOs to generate ∆Trp-containing derivatives [ 14 ]. In spite of the larger number of CDOs tested in this study and their distribution on the phylogenetic tree, we observed similar results, with few ∆Trp-containing derivatives.…”

“…1). Furthermore, incomplete data have been obtained for a fifth CDO-containing pathway encoded in Nocardiopsis prasina, with CDPS-Np synthesizing cYF and cYY and CDO-Np catalysing the tetra-dehydrogenation of these CDPs [13,14]. CDOs are comprised of two subunits of approximately 21 and 11 kDa, referred to hereafter as CDOA and CDOB, respectively.…”

“…Additionally, the group of Kanzaki reported the conversion of phenylahistine, a prenylated CDP, to its dehydro-derivative by the CDO from Streptomyces albulus KO-23, showing the acceptance of more complex CDP substrates by CDOs [18]. Recently, AlbA/B, Ndas_1146/1147, and CDO-Np were investigated for the conversion of a large set of chemically synthesized cyclodipeptides and analogues [14]. The authors used either bioconversion using recombinant Escherichia coli bacteria that overproduce CDO or in vitro conversion using a bacterial crude extract containing recombinant CDO.…”