A special lycopene ε-cyclase from Chromochloris zofingiensis ATCC 30412 with multiple catalytic functions

The cyclization of lycopene catalyzed by lycopene cyclases (LCYs) represents a pivotal step in forming α-branch or β-branch carotenoids. However, the catalytic functions of LCYs are species-specific. In this study, the LCYs of Chromochloris zofingiensis ATCC 30412, including lycopene ε-cyclase (CzLCYE) and lycopene β-cyclase (CzLCYB), were characterized by sequence alignment, subcellular localization, and function identification. The sequence alignment revealed the presence of several conserved motifs in CzLCYE and CzLCYB, exhibiting high similarity to the LCYs of other organisms. Subcellular localization experiments illustrated the chloroplast localization of these two enzymes. Functional complementation assays in Escherichia coli demonstrated that CzLCYB exhibited merely β-cyclase activity. Interestingly, CzLCYE displayed strong ε-monocyclase activity, moderate β-monocyclase activity, and weak β, β-bicyclase activity, which are rarely found in other organisms. Further, the structures of CzLCYE complexes were built to identify the essential amino acid residues within the substrate-binding pocket. Site-directed mutagenesis experiments revealed the significant roles of TYR140, PHE321, and THR337 in maintaining CzLCYE activity as well as LEU236, PHE245, PHE405, and LEU488 in β-ring formation. However, the mutation of THR205 to ALA enhanced β, β-bicyclase activity. These findings reveal the special catalytic functions of CzLCYE, contributing to a deeper understanding of carotenoid biosynthesis in C. zofingiensis.