Wu Nan, Kobayashi Naohiro, Kuroda Yutaka, Yamazaki Toshio
College of Food and Bioengineering, Zhengzhou University of Light Industry, 136 Kexue Road, Zhengzhou, 450001 People's Republic of China.
RIKEN Center for Biosystems Dynamics Research, RSC, RIKEN, 1-7-22 Suehiro-Cho, Tsurumi-Ku, Yokohama, Kanagawa 230-0045 Japan.
Biophys Rev. 2022 Dec 14;14(6):1513-1520. doi: 10.1007/s12551-022-01025-6. eCollection 2022 Dec.
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 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 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.
The online version contains supplementary material available at 10.1007/s12551-022-01025-6.
腔肠素酶（GLuc，18.2 kDa；168个残基）是一种海洋桡足类腔肠素酶，在氧化腔肠素（CTZ）时发出明亮的蓝光。GLuc是一种小型腔肠素酶，作为一种潜在的报告蛋白备受关注。然而，与萤火虫荧光素酶和已经得到充分表征并广泛应用的荧光素酶相比，GLuc的结构和生物物理研究进展缓慢。在这里，我们从结构角度综述了GLuc生物发光的生物物理和突变研究，特别是鉴于其最近的核磁共振溶液结构，其中两个同源的连续重复序列形成两个反平行束，每个束由四个螺旋组成，抓住一个短的N端螺旋。此外，一个被归类为内在无序区域的长环将两个束分开，形成一个疏水口袋的一侧，这个口袋很可能是结合/催化位点。我们将核磁共振确定的结构与最近的AlphaFold2预测结果进行了比较。总体而言，AlphaFold2结构与溶液结构一致；然而，令人惊讶的是，它揭示了一种可能的替代构象，其中N端螺旋被C端尾巴中新形成的螺旋所取代，而该螺旋在核磁共振结构中是未折叠的。此外，我们讨论了先前突变分析的结果，重点是通过化学位移扰动分析和使用核磁共振和AlphaFold2结构进行的分子动力学模拟确定的假定催化核心。特别是，我们讨论了可能的构象变化和疏水口袋在GLuc活性中的作用。总体而言，讨论指向了GLuc意想不到的和不寻常的特征，这些特征似乎比传统酶更加灵活，从而导致了一种独特的催化模式来实现CTZ的氧化脱羧。
在线版本包含可在10.1007/s12551-022-01025-6获取的补充材料。
