| 耿泽云,段贺,赵晨莹,等.深海冷泉来源诺卡氏菌OUCLQ19-35中环二肽氧化酶NczA_B的体外功能研究[J].中国海洋药物,2025,(6):-. |
| 深海冷泉来源诺卡氏菌OUCLQ19-35中环二肽氧化酶NczA_B的体外功能研究 |
| In vitro biochemical characterization of cyclodipeptide oxidase NczA_B from Nocardiopsis sp. OUCLQ19-35 |
| 投稿时间：2024-05-14 修订日期：2024-07-09 |
| DOI： |
| 中文关键词: 深海冷泉 二酮哌嗪类化合物 环二肽氧化酶 环二肽合酶基因簇 |
| English Keywords:deep-sea derived diketopiperazines cyclodipeptide oxidases cyclodipeptide synthase biosynthesis gene cluster |
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| 中文摘要: |
| 目的 对深海冷泉来源的诺卡氏菌Nocardiopsis sp. OUCLQ19-35中的环二肽氧化酶NczA_B进行体外生化功能的探究,为生物改造二酮哌嗪类化合物提供酶元件。方法 对ncz基因簇进行生物信息学分析,预测NczA_B催化底物及生化功能,通过分子克隆技术获得环二肽氧化酶基因(nczA_B),构建蛋白表达载体pET28a::nczA_B,在大肠杆菌BL21(DE3)中进行可溶性表达；体外建立酶促反应体系,通过高效液相色谱法(HPLC)分析酶反应结果,并且通过MS/MS手段对酶反应产物进行结构鉴定。结果 NczA_B可催化cyclo(L-Phe-L-Tyr)(cFY)、cyclo(L-Phe-L-Phe)(cFF)进行Cα-Cβ脱氢反应,生成氧化脱氢产物；进一步对其进行底物宽泛性测试,发现其可催化cyclo(L-Tyr-L-Tyr)(cYY)、cyclo(L-Trp-L-Leu)(cWL)、cyclo(L-Trp-L-Pro)(cWP),无法识别cyclo(L-Trp-L-Val)(cWV)。结论 本研究通过体外生化反应,发现NczA_B是具有一定的底物宽泛性的环二肽氧化酶,为后续合成一系列脱氢的二酮哌嗪类化合物提供了一个具有较强底物宽泛性的酶元件。 |
| English Summary: |
| Objective The aim of this study was to investigate the in vitro biochemical function of cyclodipeptide oxidase NczA_B from Nocardiopsis sp. OUCLQ19-35, isolated from deep-sea cold seeps, to provide enzymatic components for the biotransformation of diketopiperazine compounds. Methods The ncz gene cluster was subjected to bioinformatics analysis to predict the catalytic substrates and biochemical functions of NczA_B. The cyclodipeptide oxidase gene (nczA_B) was obtained through molecular cloning techniques, and the protein expression vector pET28a::nczA_B was constructed for soluble expression in Escherichia coli BL21(DE3). An enzyme-catalyzed reaction system was established in vitro, and the reaction results were analyzed by high-performance liquid chromatography (HPLC). Furthermore, the enzyme reaction products were structurally identified using MS/MS. Results NczA_B could catalyze the Cα-Cβ dehydrogenation reactions of cyclo(L-Phe-L-Tyr)(cFY) and cyclo(L-Phe-L-Phe)(cFF) to generate oxidative dehydrogenation products. Further substrate specificity testing revealed that it could catalyze cyclo(L-Tyr-L-Tyr)(cYY), cyclo(L-Trp-L-Leu)(cWL), and cyclo(L-Trp-L-Pro)(cWP) but could not recognize cyclo(L-Trp-L-Val)(cWV). Conclusion This study identified NczA_B as a cyclodipeptide oxidase with certain substrate promiscuity through in vitro biochemical reactions, providing an enzymatic component with broad substrate specificity for the synthesis of a series of dehydrogenated diketopiperazine compounds. |
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