An integrated engineering strategy enhances expression and activity of Pseudonocardia thermophila nitrile hydratase in Escherichia coli.
Nitrile hydratase is a key enzyme for nitrile-to-amide hydration under mild conditions, yet its application is limited by low heterologous expression, poor solubility, and suboptimal catalysis. Here, we present an integrated strategy to enhance expression, assembly, and function of Pseudonocardia thermophila NHase (PtNHase) in Escherichia coli, and dissect its mechanism via kinetic analysis. Codon optimization improved total whole-cell activity by 99% at 36 °C, likely through enhanced translational efficiency and co-translational folding at elevated temperatures. Replacing the native Shine-Dalgarno sequence with a strong RBS increased α-subunit expression 1.8-fold and boosted total whole-cell activity by 215%, outperforming solubilization tag and subunit fusion. Structure-guided saturation mutagenesis identified βTrp72 as a key specificity determinant. The βW72F variant increased apparent activity toward 3-cyanopyridine by 45% while retaining wild-type acrylonitrile activity. However, kinetic analysis revealed that βW72F has lower intrinsic catalytic efficiency (kcat/Km 10.8 vs. 30.9 mM-1·s-1), and its apparent gain arises from relieved substrate inhibition at high substrate concentrations (Kᵢ of βW72F increased from 306.1 to 688.4 mM), rather than from enhanced intrinsic rate. This mutation exhibits a trade-off: enhanced activity and tolerance toward 3-cyanopyridine but reduced thermostability and nicotinamide tolerance. Computational analyses suggested that βW72F expands the substrate tunnel entrance (bottleneck radius 0.92 → 1.32 Å), optimizes CoN coordination (5.8 → 4.9 Å), but compromises thermostability due to increased backbone flexibility (RMSD 2.78 → 3.42 Å). This work establishes a high-efficiency expression system for PtNHase and highlights that apparent activity gain does not necessarily equate to improved intrinsic catalytic efficiency, providing a framework for engineering complex metalloenzymes.