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 C–C 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 2,5-diketopiperazine 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 using2H-labeled substrates; Product identification also by interpretation of MS2fragmentation pattern.

Highlights

  • Cyclodipeptides (CDPs) containing a 2,5-diketopiperazine (DKP) backbone represent an important group of precursors in drug development (Borthwick 2012)

  • DKP backbone through condensation of two amino acids can be catalyzed by non-ribosomal peptide synthetases (NRPSs) or cyclodipeptide synthases (CDPSs) (Borgman et al 2019; Canu et al 2020)

  • Sequence comparison revealed that Cyclodipeptide oxidases (CDOs)-NpA shares a sequence identity of 41% with AlbA and 68% with Ndas_1146 on the amino acid level

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Summary

Introduction

Cyclodipeptides (CDPs) containing a 2,5-diketopiperazine (DKP) backbone represent an important group of precursors in drug development (Borthwick 2012) They are mainly produced by microorganisms in various environments and are known to exhibit diverse biological and pharmaceutical activities (Borthwick 2012; Huang et al 2010). DKP backbone through condensation of two amino acids can be catalyzed by non-ribosomal peptide synthetases (NRPSs) or cyclodipeptide synthases (CDPSs) (Borgman et al 2019; Canu et al 2020) These cyclodipeptides (CDPs) can undergo several tailoring modifications such as methylation, hydroxylation, prenylation, dimerization/coupling, and dehydrogenation, which can enhance their biological and pharmacological activities (Borgman et al 2019; Canu et al 2020; Giessen and Marahiel 2014; Li et al 2014; Yu et al 2018)

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