Crystal Structure of 4-Hydroxyphenylpyruvate Dioxygenase in Complex with Substrate Reveals a New Starting Point for Herbicide Discovery

Abstract

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a promising target for drug and pesticide discovery. The unknown binding mode of substrate is still a big challenge for the understanding of enzymatic reaction mechanism and novel HPPD inhibitor design. Herein, we determined the first crystal structure of <i>Arabidopsis thaliana</i> HPPD (<i>At</i>HPPD) in complex with its natural substrate (HPPA) at a resolution of 2.80 Å. Then, combination of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations confirmed that HPPA takes keto rather than enol form inside the HPPD active pocket. Subsequent site-directed mutagenesis and kinetic analysis further showed that residues (Phe424, Asn423, Glu394, Gln307, Asn282, and Ser267) played important roles in substrate binding and catalytic cycle. Structural comparison between HPPA-<i>At</i>HPPD and holo-<i>At</i>HPPD revealed that Gln293 underwent a remarkable rotation upon the HPPA binding and formed H-bond network of Ser267-Asn282-Gln307-Gln293, resulting in the transformation of HPPD from an inactive state to active state. Finally, taking the conformation change of Gln293 as a target, we proposed a new strategy of blocking the transformation of HPPD from inactive state to active state to design a novel inhibitor with <i>K</i> _i value of 24.10 nM towards <i>At</i>HPPD. The inhibitor has entered into industry development as the first selective herbicide used for the weed control in sorghum field. The crystal structure of <i>At</i>HPPD in complex with the inhibitor (2.40 Å) confirmed the rationality of the design strategy. We believe that the present work provides a new starting point for the understanding of enzymatic reaction mechanism and the design of next generation HPPD inhibitors.