title: The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase.
abstract: 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a non-haem iron(II)-dependent oxygenase that catalyses the conversion of 4-hydroxyphenylpyruvate (HPP) to homogentisate (HG). In the active site, a strictly conserved 2-His-1-Glu facial triad co-ordinates the iron ready for catalysis. Substitution of these residues resulted in about a 10-fold decrease in the metal binding affinity, as measured by isothermal titration calorimetry, and a large reduction in enzyme catalytic efficiencies. The present study revealed the vital role of the ligand Glu(349) in enzyme function. Replacing this residue with alanine resulted in loss of activity. The E349G variant retained 5% activity for the coupled reaction, suggesting that co-ordinating water may be able to support activation of the trans-bound dioxygen upon substrate binding. The reaction catalysed by the H183A variant was fully uncoupled. H183A variant catalytic activity resulted in protein cleavage between Ile(267) and Ala(268) and the production of an N-terminal fragment. The H266A variant was able to produce 4-hydroxyphenylacetate (HPA), demonstrating that decarboxylation had occurred but that there was no subsequent product formation. Structural modelling of the variant enzyme with bound dioxygen revealed the rearrangement of the co-ordination environment and the dynamic behaviour of bound dioxygen in the H266A and H183A variants respectively. These models suggest that the residues regulate the geometry of the reactive oxygen intermediate during the oxidation reaction. The mutagenesis and structural simulation studies demonstrate the critical and unique role of each ligand in the function of HPPD, and which correlates with their respective co-ordination position.
authors.affiliation: Pharmacy Division, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Hsiu-Chen
authors.initials: HC
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Chia-Pei
authors.initials: CP
authors.lastname: Shen
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Yi-Tong
authors.initials: YT
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Sung-Jai
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Medicinal Chemistry, Department of Pharmacy & Pharmacology, Claverton Down, University of Bath, Bath, U.K.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Lloyd
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan hjlee@mail.ndmctsgh.edu.tw.
authors.forename: Hwei-Jen
authors.initials: HJ
authors.lastname: Lee
mesh_terms: D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D020125:Mutation, Missense
chemical_list: D008024:Ligands; D007501:Iron; D015111:4-Hydroxyphenylpyruvate Dioxygenase
keywords: 2-His-1-carboxylate facial triad; 4-hydroxy-phenylpyruvate dioxygenase; iron-binding ligands; metal-binding motif; non-haem Fe(II)-dependent oxygenase