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

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          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 Arabidopsis thaliana HPPD ( AtHPPD) 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- 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. The inhibitor has entered into industry development as the first selective herbicide used for the weed control in sorghum field. The crystal structure of AtHPPD 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.

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          Geometric and electronic structure/function correlations in non-heme iron enzymes.

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            The behavior and significance of slow-binding enzyme inhibitors.

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              4-Hydroxyphenylpyruvate dioxygenase.

              R. Moran (2005)
              4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an Fe(II)-dependent, non-heme oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. This reaction involves decarboxylation, substituent migration and aromatic oxygenation in a single catalytic cycle. HPPD is a member of the alpha-keto acid dependent oxygenases that typically require an alpha-keto acid (almost exclusively alpha-ketoglutarate) and molecular oxygen to either oxygenate or oxidize a third molecule. As an exception in this class of enzymes HPPD has only two substrates, does not use alpha-ketoglutarate, and incorporates both atoms of dioxygen into the aromatic product, homogentisate. The tertiary structure of the enzyme would suggest that its mechanism converged with that of other alpha-keto acid enzymes from an extradiol dioxygenase progenitor. The transformation catalyzed by HPPD has both agricultural and therapeutic significance. HPPD catalyzes the second step in the pathway for the catabolism of tyrosine, that is common to essentially all aerobic forms of life. In plants this pathway has an anabolic branch from homogentisate that forms essential isoprenoid redox cofactors such as plastoquinone and tocopherol. Naturally occurring multi-ketone molecules act as allelopathic agents by inhibiting HPPD and preventing the production of homogentisate and hence required redox cofactors. This has been the basis for the development of a range of very effective herbicides that are currently used commercially. In humans, deficiencies of specific enzymes of the tyrosine catabolism pathway give rise to a number of severe metabolic disorders. Interestingly, HPPD inhibitor/herbicide molecules act also as therapeutic agents for a number of debilitating and lethal inborn defects in tyrosine catabolism by preventing the accumulation of toxic metabolites.
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                Author and article information

                Contributors
                Journal
                Res (Wash D C)
                Res (Wash D C)
                RESEARCH
                Research : a Science Partner Journal
                AAAS
                2639-5274
                2019
                8 July 2019
                : 2019
                : 2602414
                Affiliations
                1Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint Research Center for Intelligent Biosensor Technology and Health, College of Chemistry, Chemical Biology Center, Central China Normal University, Wuhan 430079, China
                2MOE Key Laboratory of Protein Sciences, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
                3College of Chemistry and Material Science, South-Central University for Nationalities, Wuhan 430074, China
                4Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, USA
                5Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, China
                Article
                10.34133/2019/2602414
                6750108
                31549053
                50792ff5-6612-425b-8630-f41c7af90700
                Copyright © 2019 Hong-Yan Lin et al.

                Exclusive licensee Science and Technology Review Publishing House. Distributed under a Creative Commons Attribution License (CC BY 4.0).

                History
                : 19 March 2019
                : 10 June 2019
                Funding
                Funded by: National Key Research and Development Program of China
                Award ID: 2017YFD0200500
                Funded by: National Natural Science Foundation of China
                Award ID: 21837001
                Award ID: 21672079
                Funded by: Hubei Province Natural Science Foundation
                Award ID: 2018CFA072
                Funded by: Central China Normal University
                Award ID: CCNU18ZDPY01
                Award ID: CCNU18TS007
                Categories
                Research Article

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