The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase

Huang, Chih-Wei L.; Liu, Hsiu-Chen; Shen, Chia-Pei; Chen, Yi-Tong; Lee, Sung-Jai; Lloyd, Matthew D.; Lee, Hwei-Jen

doi:10.1042/bcj20160146

Cited by 14 publications

(21 citation statements)

References 52 publications

(74 reference statements)

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“…Extensive crystallographic and spectroscopic studies have shown that the metal ion is sixfold coordinated with the 2-His-1-Glu facial triad (His226 and His308 and one Glu394) and three water molecules at the active site [10, 30, 39]. Similarly, in HPPD-HPPA complex, the α -ketoglutarate moiety of HPPA in a bidentate interaction results in an octahedral coordination geometry involving the facial triad (Figure 1(b)).…”

Section: Resultsmentioning

confidence: 99%

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

Lin

Chen

et al. 2019

Research

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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 Ki 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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Section: Resultsmentioning

confidence: 99%

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

Lin

Chen

et al. 2019

Research

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“…HPD acts upstream of FAH in the tyrosine catabolism pathway and Hpd disruption ameliorates HT-I symptoms by preventing the toxic metabolite build-up that results from loss of FAH. Structural analyses of HPD reveal that the catalytic domain of the HPD enzyme is located at the C-terminus of the enzyme and encoded by exon 13 and 14 [ 57 ]. Thus, frameshift-inducing indels upstream of exon 13 should render the enzyme inactive.…”

Section: Discussionmentioning

confidence: 99%

All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo

et al. 2018

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BackgroundClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.ResultsHere, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail-vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded > 35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo.ConclusionsOur findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1515-0) contains supplementary material, which is available to authorized users.

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“…Structural analyses of HPD reveal that the catalytic domain of HPD enzyme is located at the Cterminus of the enzyme and encoded by exon 13 and 14 (54). Thus, frameshift-inducing indels upstream of exon 13 should render the enzyme inactive.…”

Section: Pathway Reprogramingmentioning

confidence: 99%

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Ibraheim¹,

Song²,

Mir³

et al. 2018

Preprint

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Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. Here we demonstrate that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.3 SignificanceWe and others have shown that NmeCas9 is an efficient Cas9 nuclease with a high degree of specificity in cultured human cells. NmeCas9 has a unique PAM that expands the targeting range of Cas9-based genome editing. Since NmeCas9 is more compact than SpyCas9, its delivery with its guide-RNA in a single recombinant adeno-associated virus becomes a viable option. Furthermore, NmeCas9 has an unusually low off-target profile. These characteristics make NmeCas9 an ideal candidate for in vivo genome editing, including therapeutic gene knockouts, as we demonstrate in the mouse liver. We anticipate that successful in vivo delivery of this accurate and effective Cas9 can advance therapeutic editing in humans.

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The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase

Cited by 14 publications

References 52 publications

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

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

All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

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