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

4-Hydroxyphenylpyruvate dioxygenase(HPPD)is a promising target for drug and pesticide discovery.Te 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 frst crystal structure of Arabidopsis thaliana HPPD(AtHPPD)in complex with its natural substrate(HPPA)at a resolution of 2.80˚A.Ten,combination of hybrid quantum mechanics/molecular mechanics(QM/MM)calculations confrmed 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-AtHPPD and holo-AtHPPD 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 K_(i) value of 24.10 nM towards AtHPPD.Te inhibitor has entered into industry development as the frst selective herbicide used for the weed control in sorghum feld.Te crystal structure of AtHPPD in complex with the inhibitor(2.40˚A)confrmed 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.