Biophysical analysis of Gaussia Luciferase bioluminescence mechanisms using a non-oxidizable coelenterazine.

Abstract

Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but Glucs fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine, a non-oxidizable coelenterazine analog, as a probe to investigate its binding mode to GLuc. Interestingly, the biochemical studies of GLuc inhibition by azacoelenterazine also led us to find that salt, and monovalent anions, are required for GLucs bioluminescence, which seems reasonable for a sea-dwelling creature. The NMR-based investigation, using chemical shift perturbations monitored by 15NH-HSQC, suggested that CTZ binds to residue in or near the hydrophobic cavity. Of note is that these NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.