Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera  

作　　者：Yuetong Yu Yan Liu Gangqiang Dong JinZhu Jiang Liang Leng XianJu Liu Jun Zhang An Liu Sha Chen 

机构地区：[1]Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,No.16,Nanxiaojie,Dongzhimennei,Beijing 100700,China [2]Amway(China)Botanical R&D Centre,Wuxi 214115,China

出　　处：《Horticulture Research》2023年第2期241-251,共11页园艺研究（英文）

基　　金：supported by the National Natural Science Foundation of China(32170388);the Scientific and Technological Innovation project of China Academy of Chinese Medical Sciences(CACMS Innovation Fund CI2021A04108,CI2021A04515);the Fundamental Research Funds for the Central Public Welfare Research Institutes of China(ZZ13-YQ-057,ZXKT21006)。

摘　　要：Lotus(Nelumbo nucifera),an ancient aquatic plant,possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids(BIAs).However,only few genes and enzymes involved in BIA biosynthesis in N.nucifera have been isolated and characterized.In the present study we identified the regiopromiscuity of an O-methyltransferase,designated NnOMT6,isolated from N.nucifera;NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O,aporphine skeleton 6-O,phenylpropanoid 3-O,and protoberberine 2-O.We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation.Verification using site-directed mutagenesis revealed that residues D316,N130,L135,N176A,D269,and E328 were critical for BIA O-methyltransferase activities;furthermore,N323A,a mutant of NnOMT6,demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6.To the best of our knowledge,this is the first study to report the O-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in N.nucifera.The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen O-methyltransferase activity in plants.

关 键 词：BENZYL RESIDUES SKELETON 

分 类 号：S682.32[农业科学—观赏园艺]