Padi Neo, Akumadu Blessing Oluebube, Faerch Olga, Aloke Chinyere, Meyer Vanessa, Achilonu Ikechukwu
Protein Structure-Function and Research Unit, School of Molecular and Cell Biology, Faculty of Science, University of the Witwatersrand, Braamfontein, Johannesburg 2050, South Africa.
Functional Genomics and Immunogenetics Laboratory, School of Molecular and Cell Biology, Faculty of Science, University of the Witwatersrand, Braamfontein, Johannesburg 2050, South Africa.
Biomolecules. 2021 Dec 7;11(12):1844. doi: 10.3390/biom11121844.
Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related and 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa / GST (Sbh26GST) to understand structure-function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.
谷胱甘肽转移酶（GSTs）是血吸虫中的主要解毒酶。这些寄生酶在药物治疗期间往往会上调，其中 是主要感染人类的物种之一。在任何数据库中，都缺乏与 和 26-kDa GST 同工型密切相关的完整序列信息。因此，我们设计了一种伪 26-kDa / GST（Sbh26GST），以了解其结构 - 功能关系以及对选定潜在配体的配体结合蛋白活性。Sbh26GST 作为 MBP 融合蛋白在 中过表达，纯化至同质，并催化 1 - 氯 - 2,4 - 二硝基苯 - 谷胱甘肽（CDNB - GSH）结合活性，比活性为 13 μmol/分钟/毫克。在溴磺酚酞（BSP）存在下，该活性降低了约 95%，其 IC 为 27 μM。此外，酶动力学表明 BSP 相对于 GSH 起非竞争性抑制剂的作用。光谱研究证实 Sbh26GST 采用典型的 GST 结构，主要为α - 螺旋结构。进一步的外在 8 - 苯胺基 - 1 - 萘磺酸盐（ANS）光谱表明，BSP、吡喹酮（PZQ）和青蒿素（ART）可能优先结合在二聚体界面或靠近酶的疏水底物结合位点处。Sbh26GST - BSP 相互作用是由焓和熵驱动的，化学计量比为每个 Sbh26GST 二聚体一个 BSP 分子。在 BSP 和 GSH 存在下，酶的稳定性似乎增强。诱导契合配体对接证实了光谱、热力学和分子建模结果。总之，BSP 是 Sbh26GST 的有效抑制剂，有可能被合理地用作血吸虫病的治疗药物。
