Bioluminescence of (R)-Cypridina Luciferin with Cypridina Luciferase

Author:

Kanie Shusei1ORCID,Wu Chun2,Kihira Kiyohito3ORCID,Yasuno Rie4ORCID,Mitani Yasuo1,Ohmiya Yoshihiro25ORCID

Affiliation:

1. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hokkaido Center, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan

2. Biomedical Research Institute, AIST, Kansai Center, 1-8-31 Midorigaoka, Ikeda 563-8577, Japan

3. Japan Aerospace Exploration Agency (JAXA), Tsukuba Space Center, 2-1-1 Sengen, Tsukuba 305-8505, Japan

4. Cellular and Molecular Biotechnology Research Institute, AIST, Tsukuba Center, 1-1-1 Higashi, Tsukuba 305-8566, Japan

5. Department of Biomedical Engineering, Osaka Institute of Technology (OIT), 5-16-1 Ohmiya, Asahi-ku, Osaka 535-8585, Japan

Abstract

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 Km 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.

Funder

the Northern Advancement Center for Science and Technology of Hokkaido, Japan

the Ministry of Education, Culture, Sprots, Science and Technology

JSPS KAKENHI

Publisher

MDPI AG

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