斑鸠霉素氧化酶CftA体外生物活性研究

In vitro biochemical characterization of ikarugamycin oxidase CftA

摘要:

[背景] 斑鸠霉素及其氧化衍生物属于多环特特拉姆酸大环内酰胺类化合物，具有良好的生物活性，挖掘更多新颖斑鸠霉素类似物具有重要意义。细胞色素P450氧化酶CftA被认为能催化斑鸠霉素的氧化反应，但未有相关体外实验数据的报道。[目的] 通过体外生化实验鉴定氧化酶CftA的功能，并探索其催化斑鸠霉素氧化反应的机制。[方法] 利用基因合成的方法直接克隆斑鸠霉素氧化酶基因cftA，于大肠杆菌中诱导表达后，纯化蛋白进行体外酶反应，利用高效液相色谱与高分辨质谱联用技术鉴定酶反应产物。[结果] 在体外，氧化酶CftA催化斑鸠霉素生成一个新的氧化衍生物hydroxyikarugamycin D和一个已知氧化衍生物clifednamide A。[结论] 进行了细胞色素P450氧化酶CftA的体外生化研究，证实其能特异性催化斑鸠霉素C29位的两步氧化反应，为进一步探索斑鸠霉素P450氧化酶的催化机制，以及通过生物酶催化拓展斑鸠霉素类多环特特拉姆酸大环内酰胺化合物的结构多样性奠定了基础。

Abstract:

[Background] Polycyclic tetramate macrolactams (PoTeMs), especially ikarugamycin and its analogs, display a wide range of antifungal, antibacterial, and cytotoxic activities. The cytochrome P450 CftA was shown to be an ikarugamycin oxidase according to the in vivo experiments, however, no in vitro evidence was available. [Objective] To study the biochemical function of ikarugamycin oxidase CftA. [Methods] CftA was purified by Ni-NTA affinity chromatography after the overexpression of synthetic gene cftA in Escherichia coli BL21(DE3), and was biochemically characterized by in vitro enzyme reaction and identification of reaction products through HPLC-HR-ESI-MS/MS. [Results] A new ikarugamycin derivative named hydroxyikarugamycin D and a known ikarugamycin derivative, clifednamide A, were observed after incubating CftA and ikarugamycin. [Conclusion] The in vitro evidence was provided to support that tandem oxidations on ikarugamycin was carried out by CftA at C29. This result paved the way for further researches to study the mechanism of ikarugamcin oxidases and the utilization to develop novel ikarugamycin type of PoTeMs.