Pharmaceutically active compounds are an important category of emerging pollutants, and their biological transformation processes in the environment are crucial for understanding and evaluating the migration, transformation, and environmental fate of emerging pollutants. The cytochrome P450 105 enzyme family has been proven to play an important role in the degradation of exogenous environmental pollutants. However, its thermostability and catalytic activity still need to be improved to better adapt to complex environmental conditions. This work elucidates the key mechanisms and important residues of the degradation reaction through multiple computational strategies, establishes a mutation library, and obtains 21 single-point mutation designs. Experimental verification showed that 16 single mutants had enhanced thermostability, with the R89F and L197Y mutants showing the highest increases in thermostability at 135 and 119% relative to the wild-type enzyme, respectively. Additionally, as a result of the higher specific activity of D390Q, it was selected for combination mutagenesis, ultimately resulting in three combination mutants (R89F/L197Y, R89F/D390Q, and R89F/L197Y/D390Q) with enhanced thermostability and catalytic activity. This study provides a modification approach for constructing efficient enzyme variants through semirational design and can contribute to the development of control technologies for emerging pollutants.

中文翻译：

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增强细胞色素 P450 105D7 降解药物活性化合物双氯芬酸的热稳定性和催化活性的半理性设计策略

药物活性化合物是新兴污染物的重要类别，其在环境中的生物转化过程对于理解和评估新兴污染物的迁移、转化和环境归趋至关重要。细胞色素P450 105酶家族已被证明在外源环境污染物的降解中发挥重要作用。但其热稳定性和催化活性仍需提高，以更好地适应复杂的环境条件。该工作通过多种计算策略阐明了降解反应的关键机制和重要残基，建立了突变库，获得了21个单点突变设计。实验验证表明，16个单突变体的热稳定性得到增强，其中R89F和L197Y突变体相对于野生型酶的热稳定性提高最高，分别为135%和119%。此外，由于D390Q具有较高的比活性，因此选择它进行组合诱变，最终产生了三个具有增强的热稳定性和催化活性的组合突变体（R89F/L197Y、R89F/D390Q和R89F/L197Y/D390Q）。这项研究提供了一种通过半理性设计构建高效酶变体的修饰方法，有助于新兴污染物控制技术的发展。