酶是天然存在的高效催化剂,长期的进化过程产生了诸多的同源酶,同源酶的序列相似高却有不同的功能,而导致功能差异的原因也倍受关注。类胡萝卜素合成过程中,番茄红素β-环化酶(lycopene β-cyclase,LCYB)和新黄质合酶(neoxanthin synthase,NSY)序列高度相似但对番茄红素的环化功能不同,且并未有研究报道导致环化差异的原因。因此该研究选用氨基酸序列相似性为88.2%的枸杞来源LyLCYB和番茄来源SoNSY为研究对象,通过将基因soNSY的片段替换为lyLCYB对应位置的片段构建不同的嵌合体基因,并且利用定点突变策略构建差异位点的点突变蛋白,将改造后的基因在大肠杆菌中异源表达,测定其番茄红素环化活性。最终发现了219位点是导致LyLCYB和SoNSY番茄红素β-环化功能差异的关键氨基酸,为进一步探究影响其他来源的LCYB和NSY功能差异提供了理论支持。
Enzymes are naturally occurring and efficient catalysts. There are so many homologous enzymes during the long evolutionary process. The homologous enzymes have high sequence similarity but different functions, and the reasons for the functional differences are of great interest. In carotenoid synthesis,the amino acid sequences of lycopene β-cyclase (LCYB) and neoxanthin synthase (NSY) are highly similar but they have different functions in lycopene cyclization and there was no studies have reported the reasons for the differences in cyclization. Therefore, this study selected LyLCYB from Lycium barbarum origin and Solanum lycopersicum-derived SoNSY with 88.2% amino acid sequence similarity, and constructed different chimeric genes by replacing the fragment of soNSY with the fragment of lyLCYB at the corresponding position, and used the strategy of site-directed mutagenesis to construct point mutant proteins at different sites. Then, the modified genes were heterologous expression in Escherichia coli and their lycopene cyclization activity was determined. Finally, the research had identified the site of 219 as the key amino acid responsible for the functional differences in lycopene β-cyclization between LyLCYB and SoNSY. This study provides theoretical support for further investigation of the functional differences affecting LCYB and NSY from other sources.
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