阳春砂龙脑基二磷酸合酶关键氨基酸位点筛选及突变体的构建

Screening of key amino acid sites of AvBPPS and construction of mutants

目的对阳春砂萜类合成关键酶龙脑基二磷酸合酶(bornyl diphosphate synthase,BPPS)催化生成龙脑基二磷酸(bornyl diphosphate,BPP)的关键氨基酸位点解析,为阳春砂BPPS的功能优化并且促进其在代谢工程中的应用奠定基础。方法比较阳春砂、海南砂的BPPS (AvBPPS、A1BPPS)及已报道的其余3种植物的BPPS,发现AvBPPS催化产生BPP的比率较高。通过分子建模和底物对接、保守基序预测,以及与其他BPPS、单萜合酶序列多重比对的方法,筛选关键氨基酸位点,探索决定AvBPPS产生BPP及其较高比率BPP的关键活性位点,据此设计突变体,并用快速定点突变的方法获得突变体。结果筛选出了20个可能影响AvBPPS催化的关键位点,并根据这些位点,设计了20个AvBPPS突变体及1个A1BPPS突变体,成功构建其中17个突变体,诱导表达获得了纯化蛋白。结论综合使用多种分析方法,理性设计BPPS的突变位点,并获得了相应突变体及蛋白,为通过氨基酸定点突变探索BPPS的催化机制及功能优化提供了基础。

Objective Bornyl diphosphate synthase(BPPS) is a special monoterpene synthase which produces bornyl diphosphate(BPP). BPP is the key precursor for the medicinal terpenoids biosynthesis of Amomum villosum. The understanding of the BPPS key amino acid sites catalyzing the production of BPP in A. villosum will help to optimize the function of BPPS and improve its application in metabolic engineering. Methods The comparison of BPPSs from A. villosum, A. longiligulare(another plant source for Fructus Amomi) and three other BPPSs which have been reported showed that Av BPPS had higher ratio of BPP production than other four BPPSs. Through the methods of molecular modeling, substrate docking, prediction of conserved motifs, and multiple alignments to explore the key active sites of AvBPPS that determine the production for BPP. The method of fast Site-Directed Mutagenesis was used to obtain the mutants. Result Twenty key sites of Av BPPS were screened out and predicted, and according to these selected sites, 20 Av BPPS mutants and one Al BPPS mutant were designed, 17 mutants and their purified proteins have been successfully obtained.Conclusion Intergrated analysis methods were used to design BPPS mutants rationally in this study, which provides the basis to explore the catalytic mechanism and to optimize the function of AvBPPS.