Identification And Functional Study Of Coumarin Prenyltransferase In Clausena Lansium

Clausena lansium contains a significant amount of prenylated coumarins,carbazoles and derivatives with diverse structures,which contributes to the structural diversity and medicinal activity of this plant.None of the prenyltransferases involved in the biosynthesis pathway of prenylated aromatic compounds in C.lansium has been reported so far,which has limited the related in-depth biosynthesis study.Thus,the present study was carried out to identify and investigate the function of prenyltransferases genes in C.lansium:(1)Cloning and functional identification of coumarin prenyltransferase in C.lansiumFive prenyltransferase candidate genes were selected from C lansium transcriptome data through bioinformatics analysis,and then heterologously expressed in yeast and insect expression systems respectively,finally enzymatic function was identified through in vitro microsome catalysis.In combination with HPLC-MS analysis,it was found that the prenyltransferase ClPT3 could accept GPP as the prenyl donor and fuse the geranyl group to the C-8 position of umbelliferone(7-hydroxy coumarin).(2)Enzymatic properties of prenyltransferase CIPT3ClPT3 is the first coumarin prenyltransferase identified from C.lansium.In order to fully understand the properties of this enzyme,the optimum reaction time,optimum reaction temperature,optimum pH value,metal ion selectivity,KM value and substrate selectivity of the enzyme were comprehensively investigated in this study.The results revealed that ClPT3 was highly selective for both the prenyl donor and acceptor and specifically catalyzed the geranyl substitution reaction at the C-8 position of umbelliferone,which was dependent on the divalent metal ion,with an optimum reaction temperature of 35℃ and an optimum pH of 11.0(CAPS).The enzymatic kinetic analysis showed that the Km value was 76.5 μM and the Vmax was 124.0×104 μg·min-1·mg-1.(3)Preliminary investigation of the key residues in the active site of CIPT3ClPT3 is a strong membrane-bound protein with 8 transmembranes,and therefore its structure is difficult to determine.In order to investigate the structural basis of ClPT3,we used Alphafold 2 to predict the three-dimensional structure of ClPT3,combined with molecular docking to predict the key amino acid sites,and further constructed ClPT3 mutants by site-directed mutagenesis,and identified the active sites by enzymatic activity analysis.After several rounds of mutation experiments,the key active sites of ClPT3 catalytic reaction were successfully identified in this study.More notably,the mutant S170A catalyzed the C-8 geranylation of umbelliferone with greatly enhanced activity compared to the wild type,while N128A could alter the position preference of ClPT3 to produce prenylated umbelliferone not only at the C-8 position,but also at the C-6 position.In summary,this study identified a novel coumarin prenyltransferase gene from C.lansium,whose recombinant enzyme can specifically catalyse the umbelliferone prenylated at C-8 position.In this case,the enzymatic properties of ClPT3 were systematically analysed to understand the characteristics of ClPT3.The structure and activity relationship of ClPT3 was further investigated by combining structural modelling,molecular docking and targeted mutagenesis techniques,and the key active sites of the enzyme catalyzed by ClPT3 were identified.This thesis provides a paradigm for the discovery and functional study of plant-derived prenyltransferase.In addition,the results obtained in this study lay a solid foundation for further studies on the catalytic mechanism and biosynthetic applications of ClPT3.