Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp Oplophorus gracilorostris

Abstract

The bioluminescent reaction of Oplophorus takes place when the oxidation of coelenterazine (the luciferin) with molecular oxygen is catalyzed by Oplophorus luciferase, resulting in light of maximum intensity at 462 nm and the products CO2 and coelenteramide. Oplophorus luciferase has now been obtained in a highly purified state. Optimum luminescence occurs at pH 9 in the presence of 0.05--0.1 M NaCl at 40 degrees C, and, due to the unusual resistance of this enzyme to heat, visible luminescence occurs at temperatures above 70 degrees C when partially purified enzyme is used. The specific activity of purest preparations is 1.75 X 10(15) photons s-1 mg-1 at 23 degrees C. At pH 8.7, native luciferase has a molecular weight of approximately 130 000, apparently comprising 4 monomers of 31 000; at lower pHs, the native luciferase tends to polymerize. The quantum yield of coelenterazine is 0.34 at 22 degrees C with this enzyme. After the luminescent reaction, the spent solution is nonfluorescent, and likewise solutions of luciferase alone. When the bioluminescent reaction was carried out in the presence of 18O2, the product CO2 contained more than 50% C18O16O, supporting the dioxetane mechanism, but without ruling out the linear peroxide mechanism.

Purification and characterization of a novel thermostable luciferase from Benthosema pterotum

“…Moreover, it appears that about 30% of its maximum bioluminescence activity is retained at low (10°C) and very high (65°C) temperatures. In the bioluminescence systems of Cypridina, Latia, Chaetopterus, the relative activity decrease steeply when the temperature is raised, and become almost zero at a temperature higher than 40°C [23]. From the Arrhenius plots (inset of Fig.…”

Secretional luciferase of the luminous shrimpOplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase

“…Previously, the molecular weight of Oplophorus luciferase was reported to be 130 kDa (by gel ¢ltration) for the native protein, and 31 kDa after treatment with SDS [3]. In our recent study, the luciferase showed a molecular weight of approximately 106 kDa in gel ¢ltration, and we found that the molecule breaks down into 35 kDa and 19 kDa proteins upon sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) analysis.…”

“…Puri¢cation and amino acid sequence analysis of native Oplophorus luciferase A puri¢ed preparation of Oplophorus luciferase obtained in 1978 [3] was further puri¢ed by two steps of chromatography. The ¢rst step was by hydrophobic interaction chromatography on a column of butyl Sepharose 4 Fast Flow (Pharmacia; 0.7U3.5 cm) using 20 mM Tris^HCl, pH 8.5, eluting with decreasing concentrations of ammonium sulfate starting at 1.5 M. The second step was by gel ¢ltration on a column of Superdex 200 Prep (Pharmacia; 1U48 cm) in 20 mM Tris^HCl, pH 8.0, containing 50 mM NaCl.…”

“…The previous studies of Oplophorus luciferase reported in 1978 [3] showed various interesting properties of this enzyme, such as a high speci¢c activity (1.75U10 15 photons/s mg at 23³C), a high quantum yield (0.34 at 22³C) and optimum light emission at a high temperature (40³C). Recently, it has been found that the substrate speci¢city of Oplophorus luciferase is unexpectedly broad [8], revealing that bisdeoxycoelenterazine, an analogue of coelenterazine, is an excellent substrate comparable to coelenterazine [9].…”

“…The mechanism underlying the luminescence of Oplophorus involves the oxidation of Oplophorus luciferin (coelenterazine) with molecular oxygen, which is catalyzed by Oplophorus luciferase [2,3] as follows:…”