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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