The Structure,Function And Mechanism Of Celastrus Angulatus Max. Acyltransferase CaAT20

Terpenoids are one of the natural products with the most complex chemical structure and conformation.They have certain medicinal value and have been widely used in green pesticides,biopharmaceuticals and other industries.Celastrus angulatus Max.is an important medicinal plant,and its secondary metabolite Celangulin V has been widely used as pesticide.The biosynthesis of terpenoids such as Celangulin V requires the participation of acyltransferase and other enzymes.In this paper,experiments were designed based on an acyltransferase gene CaAT20,which was screened from the transcriptome data.Firstly,the basic bioinformatics analysis was performed.Secondly,the prokaryotic expression vector p ET28a-CaAT20 was constructed and transfected into E.coli BL21.Different types of acyl donors and acyl acceptor substrates were selected for the functional identification and in vitro enzymatic properties study of CaAT20.Finally,the method combining molecular simulation prediction and experimental verification was used to explore the mechanism of acyltransferase CaAT20.Site-directed mutagenesis of amino acid was also studied to further improve the catalytic activity of CaAT20.Bioinformatics analysis showed that CaAT20 contains the conserved motifs of HXXXD and DFGWG and belongs to the V subfamily of the BAHD family.The optimal induction conditions for protein expression were 0.4 m M IPTG at 16℃.Results of the acylation indicated that CaAT20 could acylate geraniol to geranyl acetate and geranyl benzoate.Based on the analysis of enzymatic properties,the optimum enzymatic reaction temperature of CaAT20 was determined to be 30℃,and the optimal enzymatic reaction p H was 7.5.Furthermore,the catalytic activity of CaAT20 with benzoyl-Co A as acyl donor was higher than that with acetyl-Co A as acyl donor.Results of molecular simulation prediction and experimental verification showed that HIS166,TYR367 and ARG374 are critical amino acids in the binding process of CaAT20 and geraniol.The catalytic activities of S405 A and V397 T mutants were significantly improved compared with that of the wild type.In summary,the structure,function of CaAT20 and its interaction with geraniol were studied from the aspects of experiment and molecular simulation.Our results laid the foundation for further exploring the catalytic mechanism of C.angulatus acyltransferase CaAT20 and improving the catalytic ability of CaAT20.