Directed evolution of enzymes often fails to obtain desirable variants because of the difficulty in exploring a huge sequence space. To obtain active variants from a very limited number of variants available at the laboratory scale, machine learning (ML)-guided engineering of enzymes is becoming an attractive methodology. However, as far as we know, there is no example of an ML-guided functional modification of flavin-containing monooxygenase (FMO). FMOs are known to catalyze a variety of oxidative reactions and are involved in the biosynthesis of many natural products (NPs). Therefore, it is expected that the ML-guided functional enhancement of FMO can contribute to the efficient development of NP derivatives. In this research, we focused on p-hydroxybenzoate hydroxylase (PHBH), a model FMO, and altered only four amino acid residues around the substrate binding site. ML models were trained with a small initial library covering only approximately 0.1% of the whole sequence space, and the ML-predicted second library was enriched with active variants. The variant with the highest activity in the second library was PHBH-MWNL (V47M, W185, L199N, and L210), whose activity was more than 100 times that of the wild-type PHBH. For elucidation of the mechanism of the observed activity enhancement, the crystal structure of PHBH-MWNL in complex with 4-hydroxy-3-methyl benzoic acid was determined. In the PHBH-MWNL crystal structure, the missing water molecule WAT2 was observed due to N199 hydrogen-bonding to WAT2, indicating that the L199N mutation contributed to the observed functional improvement by stabilizing the proton relay network proposed to be important in catalysis.

中文翻译：

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通过机器学习方法增强含黄素单加氧酶的功能

由于难以探索巨大的序列空间，酶的定向进化常常无法获得理想的变异体。为了从实验室规模的数量非常有限的变体中获得活性变体，机器学习 (ML) 引导的酶工程正在成为一种有吸引力的方法。然而，据我们所知，还没有机器学习引导的含黄素单加氧酶（FMO）功能修饰的例子。 FMO 已知可催化多种氧化反应，并参与许多天然产物 (NP) 的生物合成。因此，预计机器学习引导的 FMO 功能增强有助于 NP 衍生物的高效开发。在这项研究中，我们重点关注对羟基苯甲酸羟化酶 (PHBH)（一种 FMO 模型），仅改变了底物结合位点周围的四个氨基酸残基。 ML 模型使用仅覆盖整个序列空间约 0.1% 的小型初始库进行训练，而 ML 预测的第二个库则富含活跃变体。第二个文库中活性最高的变体是PHBH-MWNL（V47M、W185、L199N和L210），其活性是野生型PHBH的100倍以上。为了阐明所观察到的活性增强的机制，确定了 PHBH-MWNL 与 4-羟基-3-甲基苯甲酸复合物的晶体结构。在 PHBH-MWNL 晶体结构中，由于 N199 与 WAT2 形成氢键，观察到缺失的水分子 WAT2，这表明 L199N 突变通过稳定被认为在催化中很重要的质子中继网络，有助于观察到的功能改善。