Supplementary MaterialsESM 1: (PDF 15. ring. N-Methylation in the diketopiperazine band or prenylation from the tryptophan-containing cyclodipeptides affects the enzyme item and activity range. Tips the DKP bands tend to be catalyzed by cyclodipeptide oxidases (CDOs). This is observed for sp first. KO-2388 (Kanzaki et al. 1999) and 2?years later in (Gondry et al. 2001). The accountable enzyme includes two subunits, AlbB and AlbA, which are structured in the genome with an overlapping series of 56?bps and so are located in a nearby from the CDPS gene (Lautru et al. 2002). The next known CDO composed of the two subunits Ndas_1146 and Ndas_1147, also located together with a CDPS gene and was proven to be responsible for ,-dehydrogenation in the biosynthesis of the nocazine family (Giessen et al. 2013b). CDOs are multimeric flavoenzymes with molecular weights of more than 2000?kDa and an estimated ratio between subunit B and A?of 1:10 (Gondry et al. 2001; Lautru et al. 2002). Expression of subunit A without B did not result in dehydrogenation of Rabbit Polyclonal to PARP (Cleaved-Gly215) cyclodipeptides, indicating that both subunits are necessary for enzyme activity (Belin et al. 2012). Enzyme assays under anaerobic conditions did not lead to dehydrogenated products; thus, the reaction is oxygen-dependent and the formation of H2O2 was also reported (Giessen et al. 2013b; Gondry et al. 2001; Lautru et al. 2002). Activity testing of Mitoxantrone supplier CDOs from revealed good acceptance of aromatic amino acidCcontaining substrates, while CDPs consisting of two aliphatic amino acids were poor substrates (Giessen et al. 2013a; Gondry et al. 2001; Kanzaki et al. 2000b; Kanzaki et al. 2004). Dehydrogenation of isoprenylated showed that CDOs have the potential of accepting more complex structures than simple CDPs (Kanzaki et al. 2004). However, other modified CDPs or d-configured CDPs were not yet tested. Therefore, a systematic investigation and comparison of different CDOs toward various CDPs and derivatives will provide new insights into the catalytic features of this enzyme group, e.g., substrate and product spectra. During our investigation on CDP-forming enzymes, we identified a CDPS from (Table S2, Supplementary Information) and used them for the biotransformation of 32 CDPs (1aC3a, 5aC21a, 24aC25a, and 27aC36a, Fig. S1, Table S2). Based on the results of feeding experiments, 34 CDPs and derivatives (1aC23a, 25aC26a, 30a, 36aC37a, Mitoxantrone supplier 39aC44a, Figs.?3, S1, and S4CS7) were chosen for enzymatic conversion with crude protein extracts. The di- and tetradehydrogenated products were identified Mitoxantrone supplier by UV, NMR, and MS analyses. Open in a separate window Fig. 3 Dehydrogenation reactions of CDPs catalyzed by the three CDOs with overall product yields of more than 30% are shown. Product yields of more than 20% are highlighted in bold. Product yields were calculated by using the area under the curves of the respective extracted ion chromatograms and indicates the mean value of two independent experiments. See Fig. S1 for detailed structures Materials and methods Chemicals (37a) and (38a) were synthesized from Boc-l-Phe and Boc-l-Trp with l-[3,3-d2]-Tyr methyl ester as described previously (Jeedigunta et al. 2000; Nie et al. 2012). Compounds 42a?44a were synthesized as described in the literature (Dawidowski and Turlo 2014; Zeng et al. 2005). Preparation of prenylated CDPs using prenyltransferases has been reported before (Fan and Li 2013; Wunsch et al. 2015; Yin et al. 2013; Yin et al. 2007). All other cyclodipeptides used in this study were obtained from Bachem (Bubendorf, Switzerland) or synthesized as reported previously (Wollinsky et al. 2012; Yu et al. 2013). Stock solutions of 20?mM cyclodipeptides were prepared in DMSO. Strains and culture conditions NRRL B-16235 was kindly provided by the Agricultural Research Service Culture Collection (NRRL, Peoria, IL, USA). ATCC 11455 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultivated according to a previously published protocol (Brockmeyer and Li 2017)..