Abstracts

Luminous ostracods of the family Cypridinidae produce light through the oxidation reaction of Cypridina luciferin (CypL) catalyzed by Cypridina luciferase (CypLase). CypL is an imidazopyrazinone compound having one chiral carbon atom, and the absolute configuration of natural CypL was determined to be S by analysis using D- and L-amino acid oxidases and Cypridina hydroluciferin that was obtained by hydrogenation of natural CypL. Additionally, previous studies reported that the reaction of (S)-CypL with CypLase produced light. However, the luminescence of optically pure (R)-CypL, which is the enantiomer of (S)-CypL, with CypLase has not been investigated, and the ability of CypLase to recognize the chirality of CypL for light emission remains unclear. In this study, to confirm whether (R)-CypL functions as a luminous substrate for CypLase, we examined the luminescence of (R)-CypL, which was successfully obtained by chiral high-performance liquid chromatography (HPLC) separation of the enantiomeric mixture, with a recombinant CypLase. Our luminescence measurements demonstrated that the reaction of (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 tenfold lower than that of (S)-CypL with CypLase, but unexpectedly our kinetic analysis of CypLase suggested a higher affinity of (R)-CypL for CypLase than (S)-CypL. Furthermore, the chiral 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. Our findings will contribute to understanding the ability of CypLase to recognize the chirality of CypL for light emission and provide a new insight into the engineering of CypLase to produce light efficiently.