基于定向进化的雷公藤三萜C-29位羧化酶催化机制研究  

Study on the catalytic mechanism of triterpene C-29 carboxylases from Tripterygium wilfordii based on directed evolution

作　　者：刘攀婷 张逸风[2] 刘远 高杰 马林[1] 吴晓毅[1] 胡雅婷 苏平[2] 袁仕君 张夏楠[1] 高伟[1] LIU Pan-ting;ZHANG Yi-feng;LIU Yuan;GAO Jie;MA Lin;WU Xiao-yi;HU Ya-ting;SU Ping;YUAN Shi-jun;ZHANG Xia-nan;GAO Wei(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;National Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]首都医科大学中医药学院,北京100069 [2]道地药材品质保障与资源持续利用全国重点实验室,中国中医科学院中药资源中心,北京100700

出　　处：《药学学报》2024年第6期1883-1893,共11页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金面上项目(81973418,81974515);国家重点研发计划项目(2020YFA0908000);国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202005).

摘　　要：雷公藤红素和雷公藤内酯甲是中药雷公藤的主要三萜活性成分,具有抗肿瘤、抗炎及免疫抑制等药理活性,是雷公藤相关中药制剂发挥临床疗效的物质基础。通过解析活性成分生源途径,优化基因元件并利用“细胞工厂”异源获取将是未来“低成本、高效率”获得雷公藤中三萜活性成分的有效途径。CYP712Ks是参与雷公藤木栓烷型和齐墩果烷型三萜骨架修饰的第一个细胞色素P450酶,发挥C-29位羧基化功能,可催化木栓酮生成美登木酸,催化β-香树素生成3-表卡通酸。本研究以四条多功能TwCYP712K1/2/3/5为研究对象,利用酿酒酵母体内功能表征,明确其催化功能及底物选择偏好性;通过同源建模和分子对接,明确蛋白与底物结合空间结构,进而对蛋白活性口袋内差异氨基酸进行互相突变,阐明决定催化功能和底物结构选择的关键氨基酸。本研究共构建了63个突变体元件,解析了影响TwCYP712Ks羧基化功能的氨基酸位点,特别是揭示了影响TwCYP712K2对齐墩果烷型和木栓烷型三萜底物选择性的关键氨基酸,TwCYP712K2F127I与TwCYP712K2A227T突变体将导致产物比例发生逆转。本研究以同源蛋白比对及相互突变的方式对TwCYP712Ks四条蛋白进行半理性设计,阐明多个决定蛋白催化功能的氨基酸位点,获得系列活性提升或改变的突变体,为雷公藤三萜活性成分的生物合成提供丰富的催化元件,并初步解析了C-29位羧基化机制。Celastrol and wilforlide A are the main active triterpenoids of the traditional Chinese medicine Lei Gong Teng,which have anti-tumour,anti-inflammatory and immunosuppressive activities,and are the material basis for the clinical efficacy of Lei Gong Teng-related Chinese medicinal preparations.By analysing the biosynthetic pathway of active ingredients,optimizing genetic elements and utilizing"cell factory"to produce triterpenoids heterologously will be an effective way to obtain from Tripterygium wilfordii in the future in a"lowcost and high-efficient"manner.CYP712Ks are the first cytochrome P450s involved in the skeleton modification of friedelane-type and oleanane-type triterpenoids in T.wilfordii,and they can catalyse the generation of polpunonic acid from friedelin and 3-epi-katonic acid fromβ-amyrin by carboxylation at C-29 position.In this study,four multifunctional TwCYP712K1/2/3/5 were used to clarify the catalytic function and substrate selection preference using in vivo functional characterization in Saccharomyces cerevisiae.The spatial structure of the protein-substrate binding was clarified through homology modeling and molecular docking,and then the differential amino acids in the active pocket of the protein were mutated to clarify the crucial amino acids determining the catalytic function and the selection of substrate structure.A total of 63 mutant elements were constructed,and the amino acid sites affecting the carboxylation function of TwCYP712Ks were analyzed.In particular,the key amino acids affecting the substrate selectivity of TwCYP712K2 towards oleanane-type and friedelane-type triterpenoids were revealed and the TwCYP712K2F127I and TwCYP712K2A227T mutants would result in a reversal of the product ratio.In conclusion,four proteins of TwCYP712Ks were semi-rationally designed by homologous protein alignment and mutual mutation to elucidate multiple amino acid sites determining the catalytic function of the proteins,and a series of activity-enhancing or altering mutants were obtained,which p

关 键 词：雷公藤 CYP712K 定点突变 美登木酸 3-表卡通酸 

分 类 号：R931[医药卫生—生药学]