两种黄素蛋白对奴卡霉素化合物的催化

In vitro biochemical characterization of catalytic reaction of two flavoproteins toward nocamycin derivatives

摘要:

【背景】2,4-二酮吡咯烷类化合物奴卡霉素和替达霉素都含有C-10酮基结构，但是该结构是由两种不同的酶短链脱氢酶NcmD和黄素腺嘌呤二核苷酸(flavine adenine dinucleotide, FAD)依赖的脱氢酶TrdL分别催化形成的。然而奴卡霉素生物合成基因簇中的FAD依赖的酶NcmL是否能回补NcmD的功能，以及TrdL能否催化奴卡霉素C-10酮基的形成，尚无相关的实验证据。【目的】通过体外酶催化实验研究NcmL和TrdL对奴卡霉素II和奴卡霉素F的C-10位羟基的催化作用。【方法】通过克隆ncmL和trdL至pET-28a(+)中，然后于大肠杆菌中进行诱导表达。诱导后的蛋白经纯化后，考察了纯化的NcmL和TrdL对奴卡霉素II和奴卡霉素F的催化作用，利用高效液相色谱与高分辨质谱联用技术鉴定了酶反应产物。【结果】NcmL不能催化奴卡霉素II和奴卡霉素F的C-10位羟基的脱氢反应，TrdL能催化奴卡霉素II和奴卡霉素F的C-10位羟基脱氢，分别得到奴卡霉素I和奴卡霉素G。【结论】体外生化研究表明，NcmL不参与奴卡霉素C-10酮基的生物合成反应，TrdL具有较广的底物谱，能催化多种奴卡霉素的C-10位羟基转化为酮基。

关键词:黄素蛋白;奴卡霉素;替达霉素;丁香糖丝菌

Abstract:

[Background] The tetramic acid derivatives, nocamycins and tirandamycins, possess C-10 ketone groups, the formation of which is catalyzed by two different enzymes: a short-chain dehydrogenase NcmD and a FAD-dependent dehydrogenase TrdL, respectively. In the biosynthetic pathway of nocamycins, whether the FAD-dependent oxidase NcmL can complement the function of NcmD remains unknown. Additionally, whether TrdL can catalyze the conversion of C-10 hydroxyl group to C-10 ketone group in nocamycins is also unknown. [Objective] To characterize the catalytic roles of NcmL and TrdL in the formation of C-10 ketone groups in nocamycins by using in vitro enzymatic assays. [Methods] The trdL and ncmL genes were respectively cloned into the vector pET-28a(+), and the recombinant vectors were then overexpressed in Escherichia coli BL21. TrdL and NcmL were purified and then used for the in vitro enzymatic assays. Nocamycins F and II were used as substrates and the products generated under the catalysis of NcmL and TrdL were determined by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometer (LC-MS). [Results] NcmL did not catalyze the dehydrogenation occurred on C-10 hydroxyl group. TrdL catalyzed the hydroxyl dehydrogenation at C-10 position in nocamycins II and F, leading to the generation of nocamycins I and G, respectively. [Conclusion] In vitro biochemical assays revealed that NcmL is not involved in formation of C-10 ketone group of nocamycins. TrdL shows a broad substrate spectrum and can catalyze the formation of C-10 ketone group in nocamycins.

Key words:flavoproteins;nocamycins;tirandamycins;Saccharothrix syringae