Abstract
Gaussia princeps luciferase (GLuc 18.2 kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). GLuc is a small luciferase, attracting much attention as a potential reporter protein. However, compared to firefly and Renilla luciferases, which have been thoroughly characterized and are used in a wide range of applications, structural and biophysical studies of GLuc have been slow to appear. Here, we review the biophysical and mutational studies of GLuc's bioluminescence from a structural viewpoint, particularly in view of its recent NMR solution structure, where two homologous sequential repeats form two anti-parallel bundles, each made of four helices, grabbing a short N-terminal helix. Additionally, a long loop classified as an intrinsically disordered region separates the two bundles forming one side of a hydrophobic pocket that is most likely the binding/catalytic site. We compare the NMR-determined structure with a recent AlphaFold2 prediction. Overall, the AlphaFold2 structure was in line with the solution structure; however, it surprisingly revealed a possible, alternative conformation, where the N-terminal helix is replaced by a newly formed α helix in the C-terminal tail that is unfolded in the NMR structure. In addition, we discuss the results of previous mutational analysis focusing on a putative catalytic core identified by chemical shift perturbation analysis and molecular dynamics simulations performed using both the NMR and the AlphaFold2 structures. In particular, we discuss the role of the possible conformational change and the hydrophobic pocket in GLuc’s activity. Overall, the discussion points toward GLuc’s unexpected and unusual characteristics that appear to be much more flexible than traditional enzymes, resulting in a unique mode of catalysis to achieve CTZ oxidative decarboxylation.
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Funding
This study was supported by a JSPS Grant-in-Aid for Scientific Research (KAKENHI-18H02385) to YK and Henan Provincial Key Scientific Research Project Plan for Colleges and Universities to NW (Grant No. 23A180002).
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NW performed computational analysis. NW, NK, YK, and TY wrote the review and agree to publish it.
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Wu, N., Kobayashi, N., Kuroda, Y. et al. Reflecting on mutational and biophysical analysis of Gaussia princeps Luciferase from a structural perspective: a unique bioluminescent enzyme. Biophys Rev 14, 1513–1520 (2022). https://doi.org/10.1007/s12551-022-01025-6
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DOI: https://doi.org/10.1007/s12551-022-01025-6