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.

中文翻译：

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在二酮哌嗪环上安装CC双键的环二肽氧化酶的异同比较研究。

环二肽氧化酶（CDO）对二酮哌嗪进行脱氢作用，并在环二肽的多样化中发挥重要作用。在这项研究中，我们调查了两个已知的CDO AlbA / B和Ndas_1146 / 7，以及一个新成员CDO-Np。LC-MS监测大肠杆菌中32个环二肽的生物转化显示，富含芳香族氨基酸的环二肽的消耗量很大。仅由脂族氨基酸组成的环二肽是较差的底物。用粗酶提取物进行的体外34种底物分析和产物鉴定证明，在许多情况下，含CDO-Np的提取物可催化两个CC双键的形成。含有其他两种酶的提取物具有较低的活性，并且主要催化双脱氢反应。对于双脱氢，通常优选苯丙氨酰基或酪氨酰基位点。没有或很少接受苯二氮杂二酮和2，6-二酮哌嗪环证明了2，5-二酮哌嗪环的重要性。二酮哌嗪环上的N-甲基化或含色氨酸的环二肽的异戊烯基化会影响酶的活性和产物谱。要点：•比较三种酶的催化活性；多种环二肽及其衍生物作为底物；使用2H标记的底物测定双键形成; 产品识别也可以通过解释MS2片段化模式进行。•比较三种酶的催化活性；多种环二肽及其衍生物作为底物；使用2H标记的底物测定双键形成; 产品识别也可以通过解释MS2片段化模式进行。•比较三种酶的催化活性；多种环二肽及其衍生物作为底物；使用2H标记的底物测定双键形成; 产品识别也可以通过解释MS2片段化模式进行。