Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, Brooklyn, New York 11201, USA.
Center for Genomics and Systems Biology, New York University, New York, New York 10003, USA.
Protein Eng Des Sel. 2024 Jan 29;37. doi: 10.1093/protein/gzae015.
Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge-charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with -14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermodynamic stability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C-55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.
磷酸三酯酶（PTEs）是一类能够有效中和有机磷酸酯（OPs）的酶，OPs 是一类危险的神经毒性化学物质。由于热稳定性和溶解度低，PTEs 的催化活性较低，特别是在较高温度下。超电荷，一种通过选择性突变表面残基为带电荷残基的蛋白质工程方法，已成功用于通过促进蛋白质之间的电荷排斥来提高蛋白质的溶解度和热稳定性。我们着手通过在 Rosetta 中使用计算蛋白质超电荷算法来克服提高 PTE 对 OPs 活性的挑战。在这里，我们发现了两种超电荷的 PTE 变体，一种带负电荷（带 -14 净电荷），一种带正电荷（带 +12 净电荷），并对它们的热力学稳定性和催化活性进行了表征。我们发现带正电荷的 PTE 虽然略有但显著降低了热稳定性，这与所有温度下的催化效率损失相关，而带负电荷的 PTE 在 25°C-55°C 之间具有更高的催化活性，同时提供与亲本 PTE 相似的热稳定性特征。超电荷对催化效率的影响将为货架稳定的 PTE 的设计和酶工程的标准提供信息。
