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《Journal of Beijing University of Agriculture》 2018-03
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Steric effects of the 296 amino acid residue of Artemisia annua bisabolol synthase influence the initiation of cyclization reaction

HE Fang;HAO Qinggang;ZHAO Huifang;MU Xing;YANG Qian;LIU Huiyun;LI Zhenqiu;College of Life Sciences,Hebei University;  
【Objective】This study is to explore the mechanism of inhibition on the cyclization reaction of Artemisia annua bisabolol synthase T296 mutants.【Methods】The mutation from threonine to isoleucine at position 296 of Artemisia annua bisabolol synthase was performed using QuikChange?Multi Site-directed mutagenesis method.Mutant protein was expressed in E.coli and purified by Ni 2+affinity chromatography and the catalytic specificity was characterized by GC and GCMS.【Results】Artemisia annua bisabolol synthase T296 Imutant catalyzes(2 E,6 E)-farnesyl diphosphate mainly into farnesene,but incubating T296 Imutant with R-nerolidyl pyrophosphate resulted in the formation ofα-bisabolol,the native product of wild enzyme.【Conclusion】The volume and stereochemistry of the 296 amino acid residue side chain of Artemisia annua bisabolol synthase are key factors in the initiation of the cyclization reaction.Its steric effect inhibits the conversion of farnesyl pyrophosphate into nerolidyl pyrophosphate.
【Fund】: 国家自然科学基金项目(31570305 30900111)
【CateGory Index】: S567.219
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