摘要
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.
基金
We thank Dr.Jiang-Qing Dong for the help in structural analysis and Dr.Jun-Jun Liu for the help in QM-MM calculation.This work was funded in part by the National Key Research and Development Program of China(no.2017YFD0200500)
National Natural Science Foundation of China(nos.21837001 and 21672079)
Hubei Province Natural Science Foundation(no.2018CFA072)
the self-determined research funds of Central China Normal University(CCNU18ZDPY01 and CCNU18TS007)from the colleges’basic research and operation of MOE.We thank the Shanghai Synchrotron Radiation Facility for providing the facility support.
