Molecular characterization and structure basis of a malonyltransferase with both substrate promiscuity and catalytic regiospecificity from Cistanche tubulosa  

作　　者：Xiao Liu Yuyu Liu Xiping Xu Wenqian Huang Yaru Yan Yingxia Wang Weisheng Tian Ting Mo Xiaoxue Cui Jun Li She-Po Shi Pengfei Tu 

机构地区：[1]Modern Research Center for Traditional Chinese Medicine,Beijing Research Institute of Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China [2]Modern Research Center for Traditional Chinese Medicine,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China

出　　处：《Acta Pharmaceutica Sinica B》2024年第5期2333-2348,共16页药学学报（英文版）

基　　金：This work was financially supported by National Key Research and Development Program Special Project of Synthetic Biology(Grant No.2023YFA0914100/2023YFA0914103);National Natural Science Foundation of China(Grant No.82173922,81402809);Beijing Natural Science Foundation(Grant No.7192112);Fundamental Research Funds for the Central Universities(Grant No.2023-JYB-JBQN-054,China);Young Elite Scientists Sponsorship Program by CAST(Grant No.CACM-2018-QNRC1-02,China);State Key Laboratory of Natural and Biomimetic Drugs Foundation(Grant No.K202119,China).

摘　　要：Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply.However,the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated.This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa.It displayed unprecedented mono-and/or di-malonylation activity toward diverse glucosides with different aglycons.A“one-pot”system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides.Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides.Additionally,it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions.QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1,while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167,resulting in its high malonylation regiospecificity.Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates,emphasizing CtMaT1's preference for glucosides.Furthermore,a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained.The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation.This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives,while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

关 键 词：Plant acyltransferase Malonyltransferase Malonylated glycosides Enzymatic catalysis Enzyme promiscuity 

分 类 号：R914[医药卫生—药物化学]