Bioluminescence of ( R )-Cypridina Luciferin with Cypridina Luciferase.

Cypridina luciferin (CypL) is a marine natural product that functions as the luminous substrate for the enzyme Cypridina luciferase (CypLase). CypL has two enantiomers, ( R )- and ( S )-CypL, due to its one chiral center at the sec -butyl moiety. Previous studies reported that ( S )-CypL or racemic CypL with CypLase produced light, but the luminescence of ( R )-CypL with CypLase has not been investigated. Here, we examined the luminescence of ( R )-CypL, which had undergone chiral separation from the enantiomeric mixture, with a recombinant CypLase. Our luminescence measurements demonstrated that ( R )-CypL with CypLase produced light, indicating that ( R )-CypL must be considered as the luminous substrate for CypLase, as in the case of ( S )-CypL, rather than a competitive inhibitor for CypLase. Additionally, we found that the maximum luminescence intensity from the reaction of ( R )-CypL with CypLase was approximately 10 fold lower than that of ( S )-CypL with CypLase, but our kinetic analysis of CypLase showed that the K m value of CypLase for ( R )-CypL was approximately 3 fold lower than that for ( S )-CypL. Furthermore, the chiral high-performance liquid chromatography (HPLC) analysis of the reaction mixture of racemic CypL with CypLase showed that ( R )-CypL was consumed more slowly than ( S )-CypL. These results indicate that the turnover rate of CypLase for ( R )-CypL was lower than that for ( S )-CypL, which caused the less efficient luminescence of ( R )-CypL with CypLase.