Characterization and Catalytic Mechanism of Nitroreductases from the Novel Mesotrione-Degrading Klebsiella variicola L6

Enzyme-based degradation is crucial for mesotrione (MES) residues elimination. However, the reported enzymes are scarce, and their catalytic mechanisms underlying MES degradation remain unclear. Herein, the newly isolated Klebsiella variicola L6 was found to metabolize MES through nitro reduction and β-diketone bond oxidative cleavage. Two nitroreductases, NfsA and NfnB, which catalyze the stepwise conversion of MES into hydroxylamino and amino derivatives, were identified from strain L6. They could tolerate multiple metal ions and degrade MES within a wide range of temperatures (4–60 °C) and pH (4.0–10.0). Molecular docking and site-directed mutagenesis elucidated the mechanism of MES degradation by these enzymes. The key residues R15 and K167 (NfsA) and K14 and R107 (NfnB) are essential for their activities. Furthermore, the k cat / K m of mutants NfsA-S41A and NfnB-T41A reached 2.23- and 1.53-fold of their wild-type counterparts, respectively. This study facilitates a mechanistic understanding of nitroreductase-mediated MES degradation and provides valuable microbial and genetic resources for bioremediation.