4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for both drug and pesticide discovery. As a typical Fe(II)-dependent dioxygenase, HPPD catalyzes the complicated transformation of 4-hydroxyphenylpyruvic acid (HPPA) to homogentisic acid (HGA). The binding mode of HPPA in the catalytic pocket of HPPD is a focus of research interests. Recently, we reported the crystal structure of Arabidopsis thaliana HPPD (AtHPPD) complexed with HPPA and a cobalt ion, which was supposed to mimic the pre-reactive structure of AtHPPD-HPPA-Fe(II). Unexpectedly, the present study shows that the restored AtHPPD-HPPA-Fe(II) complex is still nonreactive toward the bound dioxygen. QM/MM and QM calculations reveal that the HPPA resists the electrophilic attacking of the bound dioxygen by the trim of its phenyl ring, and the residue Phe381 plays a key role in orienting the phenyl ring. Kinetic study on the F381A mutant reveals that the HPPD-HPPA complex observed in the crystal structure should be an intermediate of the substrate transportation instead of the pre-reactive complex. More importantly, the binding mode of the HPPA in this complex is shared with several well-known HPPD inhibitors, suggesting that these inhibitors resist the association of dioxygen (and exert their inhibitory roles) in the same way as the HPPA. The present study provides insights into the inhibition mechanism of HPPD inhibitors.

4-羟基苯基丙酮酸双加氧酶 (HPPD) 是药物和农药发现的重要目标。作为典型的 Fe(II) 依赖性双加氧酶，HPPD 催化 4-羟基苯基丙酮酸 (HPPA) 向霍黑酸 (HGA) 的复杂转化。HPPA 在 HPPD 催化口袋中的结合方式是研究热点。最近，我们报道了拟南芥HPPD ( At HPPD) 与 HPPA 和钴离子复合的晶体结构，该结构被认为是模拟At HPPD-HPPA-Fe(II)的预反应结构。出乎意料的是，目前的研究表明，恢复的AtHPPD-HPPA-Fe(II) 复合物仍然对结合的分子氧没有反应。QM/MM 和 QM 计算表明，HPPA 通过其苯环的修剪来抵抗结合双氧的亲电攻击，残基 Phe381 在苯环的定向中起关键作用。F381A 突变体的动力学研究表明，在晶体结构中观察到的 HPPD-HPPA 复合物应该是底物运输的中间体，而不是前反应复合物。更重要的是，该复合物中 HPPA 的结合模式与几种众所周知的 HPPD 抑制剂共享，表明这些抑制剂以与 HPPA 相同的方式抵抗分子氧的结合（并发挥其抑制作用）。本研究提供了对 HPPD 抑制剂抑制机制的见解。