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

作　　者：赵燕 杨松[1] 莫旭华 ZHAO Yan;YANG Song;MO Xuhua(Shandong Province Key Laboratory of Applied Mycology,School of Life Sciences,Qingdao Agricultural University,Qingdao 266109,Shandong,China)

机构地区：[1]青岛农业大学生命科学学院、山东省应用真菌重点实验室,山东青岛266109

出　　处：《微生物学通报》2023年第6期2378-2389,共12页Microbiology China

基　　金：山东省自然科学基金(ZR2020MC008)。

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。[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.

关 键 词：黄素蛋白 奴卡霉素 替达霉素 丁香糖丝菌 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] TQ426.97