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

Kyoko Takatsu; Naohiro Kobayashi; Nan Wu; Yves L Janin; Toshio Yamazaki; Yutaka Kuroda

doi:10.1016/j.bbadva.2022.100068

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

BBA Adv. 2022 Dec 23:3:100068. doi: 10.1016/j.bbadva.2022.100068. eCollection 2023.

Authors

Kyoko Takatsu¹, Naohiro Kobayashi², Nan Wu¹, Yves L Janin³, Toshio Yamazaki², Yutaka Kuroda¹

Affiliations

¹ Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
² RIKEN Center for Biosystems Dynamics Research, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.
³ Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France, Université Paris Cité.

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 GLuc's fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine (Aza-CTZ), a non-oxidizable coelenterazine (CTZ) analog, as a probe to investigate its binding mode to GLuc. While analysing GLuc's activity, we unexpectedly found that salt and monovalent anions are absolutely required for Gluc's bioluminescence, which retrospectively appears reasonable for a sea-dwelling organism. The NMR-based investigation, using chemical shift perturbations monitored by ¹⁵N-¹H HSQC, suggested that Aza-CTZ (and thus unoxidized CTZ) binds to residues in or near the hydrophobic cavity. 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.

Keywords: Azacoelenterazine; Chemical shifts; Coelenterazine; Intrinsically disordered region (IDR); NMR; SEP-tag, hydrophobic cavity; Salt concentration.