Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp....

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co²⁺-affinity chromatography. SpHPPD was found to be a monomer. The K_m and k_cat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 μM and 15.0 s^-1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC₅₀) and K_i values of 5.2 and 2.5 μM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC₅₀: 8.7 μM; K_i: 6.0 μM) and mesotrione (IC₅₀: 4.2 μM; K_i: 1.3 μM) and moderate resistance to tembotrione (IC₅₀: 2.5 μM; K_i: 1.0 μM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.