A Structural Model of Truncated Gaussia princeps Luciferase Elucidating the Crucial Catalytic Function of No.76 Arginine towards Coelenterazine Oxidation

作者：Nan Wu, Zhichao Xu, Kai-Dong Du, Shen Huang, Naohiro Kobayashi, Yutaka Kuroda, Yanhong Bai · 发表于：PLoS Computational Biology · 年份：2025 · DOI：10.1371/journal.pcbi.1012722 · 被引用次数：6 · 研究领域：bioluminescence and chemiluminescence research、Photoreceptor and optogenetics research、Molecular Communication and Nanonetworks

Gaussia Luciferase (GLuc) is a renowned reporter protein that can catalyze the oxidation of coelenterazine (CTZ) and emit a bright light signal. GLuc comprises two consecutive repeats that form the enzyme body and a central putative catalytic cavity. However, deleting the C-terminal repeat only limited reduces the activity (over 30% residual luminescence intensity detectable), despite being a key part of the cavity. How does the remaining GLuc (tGLuc) catalyze CTZ? To address this question, we built a structural model of tGLuc by removing the C-terminal repeat from the resolved structure of intact GLuc, and verified that the cavity-forming component in GLuc remains stable and provides an open-mouth cavity in tGLuc during 500 ns MD simulations in water. Docking simulation and a followed umbrella sampling analysis further revealed that the cavity on tGLuc has a high affinity for CTZ, with a binding energy of up to -114 kJ/mol. Moreover, R76, a validated activity-critical amino acid residue, resides in the cavity and forms a stable hydrogen bond with CTZ. Then, we constructed a cluster model to examine the CTZ oxidation pathway in the cavity using Density Functional Theory (DFT) calculations. The result showed that the pathway consists of four elementary reactions, with the highest Gibbs energy barrier being 65.4 kJ/mol. Both intramolecular electron transfer and the convergence of S1/S0 potential energy surfaces occurred in the last elementary reaction, which was regarded as the...