One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases

Carotenoids in the photosynthetic membranes of plants typically contain two beta -rings (e.g., beta -carotene and zeaxanthin) or one epsilon- and one beta -ring (e.g., lutein). Carotenoids with two epsilon -rings are uncommon . We reported earlier that the Arabidopsis thaliana lycopene epsilon -cycla se (LCYe) adds one epsilon -ring to the symmetrical linear substrate lycope ne, whereas the structurally related lycopene beta -cyclase (LCYb) adds two beta -rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lac tuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon -rings: lactucaxan thin. The product of the lettuce cDNA, similar in sequence to the Arabidops is LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon -carotene in a heterologous Escherichia coli system. Regio ns of the lettuce and Arabidopsis epsilon -cyclases involved in the determi nation of ring number were mapped by analysis of chimeric epsilon -cyclases constructed by using an inverse PCR approach. A single amino acid was foun d to act as a molecular switch: lettuce LCYe mutant H457L added only one ep silon -ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon -rings. An R residue in this position also yields a bi-epsilon -cyclase for both the lettuce and Arabidopsis enzymes. Constr uction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utilit y for studying membrane-associated enzymes that cannot easily be crystalliz ed or modeled to existing crystal structures.