Engineered Leghemoglobin as a High-Performance Biocatalyst for Carbene N–H Insertion: Active-Site Remodeling Unlocks Catalytic Proficiency

Leghemoglobin (LegH), a plant-derived hemoprotein, is engineered for the first time as a standalone biocatalyst for carbene N–H insertion. Through semi-rational design, the K65P mutation in the heme pocket significantly enhances catalytic efficiency. Under mild aqueous conditions (PBS buffer, 25 °C), the K65P variant achieves 92% yield in the model reaction between benzylamine and ethyl α-diazoacetate—surpassing wild-type LegH by >1.6-fold in initial reaction rate. The mutant also exhibits markedly improved thermostability. This work establishes engineered LegH as a high-performance, cofactor-free biocatalyst for C–N bond formation, providing a sustainable platform for synthesizing chiral amine derivatives. The catalytic proficiency and inherent stability of the K65P mutant demonstrate the potential of plant hemoproteins in non-natural carbene transfer reactions without requiring immobilization supports.