The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated T_m value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.

中文翻译：

---

工程化 hCGL 中不含半胱氨酸的蛋白质变体的计算引导理性设计

工程化的人胱硫醚-γ-裂解酶 (hCGL) 增强了对半胱氨酸和胱氨酸的活性，揭示了潜在的强大抗肿瘤活性。然而，半胱氨酸残基的存在有可能诱导寡聚或不正确的二硫键，这可能会降低生物药物的生物利用度。通过针对工程 hCGL 中半胱氨酸残基的精心设计过程，使用 Rosetta 和 ABACUS 进行虚拟筛选，获得了一组潜在的有益突变体。实验测量表明，大多数突变体对底物l -Cys 和 CSSC 均表现出增加的活性。此外，突变体 C109V 和 C229D 的T _m值分别增加了 8.2 和 1.8 °C。 60℃孵育80分钟后，突变体C229D仍保持较高的残留活性。出乎意料的是，突变体 C109V 对两种底物的活性均比野生型 (WT) 高约 2 倍，但在血浆中表现出令人失望的不稳定性，这表明计算设计仍需要进一步考虑。对它们的结构和分子动力学 (MD) 模拟的分析揭示了疏水相互作用、氢键和近攻击构象 (NAC) 稳定性对活性和稳定性的影响。这项研究获得了有关表现出增强的活性或耐热性的突变体的信息，并为随后的特定半胱氨酸修饰提供了宝贵的指导。