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

Abstract

<jats:p>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<jats:italic> Arabidopsis thaliana</jats:italic> HPPD (<jats:italic>At</jats:italic>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-<jats:italic>At</jats:italic>HPPD and holo-<jats:italic>At</jats:italic>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 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mrow><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant="normal">i</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math> value of 24.10 nM towards<jats:italic> At</jats:italic>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<jats:italic> At</jats:italic>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.</jats:p>

Discovery of novel arylthioacetic acid derivatives as 4‐hydroxyphenylpyruvate dioxygenase inhibitors

“…The in vitro activity of AtHPPD (Arabidopsis thaliana HPPD) was measured by coupled enzyme assay methods previously reported. 7,10,15,20 The preparation and purification of 1,2-dioxygenase (HGD) and AtHPPD were carried out according to previous methods. 15,20 HPPD inhibitory activity of the synthesized compounds were determined by a coupled enzyme method, using a coupled enzyme reaction with mesotrione as the positive control.…”

“…7,10,15,20 The preparation and purification of 1,2-dioxygenase (HGD) and AtHPPD were carried out according to previous methods. 15,20 HPPD inhibitory activity of the synthesized compounds were determined by a coupled enzyme method, using a coupled enzyme reaction with mesotrione as the positive control. Assays were performed in 96-well plates at 30°C using a UV-visible plate reader to monitor the generation of maleylacetoacetate at a wavelength of 318 nm and the inhibition constant (K i ).…”

“…13 Studies over the past decades have provided important information on interactions between HPPD enzymes and HPPD inhibitors: 1,3-dicarbonyl and aromatic moieties are an indispensable pharmacophore for potent HPPD inhibition. Many researches [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] in HPPD inhibitors indicated that modification of the aromatic moieties is an effective way to get new derivatives with excellent weed control.…”

Review on Structures of Pesticide Targets

“…In addition, the crystal structures of HPPDs from a variety of different species were systematically studied [ 62 , 63 , 64 ], and the structures in complex with NTBC [ 65 ] and a natural substrate were reported [ 66 ]. Thus, the binding mode of the substrate in the enzyme-catalyzed pocket of HPPD was revealed, which laid a solid foundation for an in-depth understanding of the mechanism of action of HPPD-inhibiting herbicides [ 67 , 68 , 69 , 70 ].…”

Atomistic Insights into the Tunable Transition from Cavitation to Crazing in Diamond Nanothread-Reinforced Polymer Composites

“…Exploring the physical mechanisms behind cavitation and crazing using a computational approach is at great challenges. At the nanoscale level, although molecular dynamics (MD) is considered as a workhorse tool to provide reliable atomic insights in composite systems [22][23][24], large-scale atomistic simulation is still essential at the expense of computational efficiency, in order to cover the entire length of cavitation and crazing sites ranging from nanometer to micrometer. At the macroscale level, despite the fact that continuum mechanics can clearly display the cavitation and crazing phenomenon and characterize the crack-growth behaviors [25], the disentanglement of polymer chains that significantly influence the cavitation-crazing transition cannot be captured.…”