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

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

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4‐Hydroxylphenylpyruvatedioxygenase (HPPD) is a non‐haem iron(II)‐dependent oxygenase that catalyzes the conversion of 4‐hydroxylphenylpyruvate (HPP) to homogentisate (HG). In the active site, a strictly conserved 2‐His‐1‐Glu facial triad coordinates 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. This study revealed the vital role of the ligand E349 in enzyme function. Substitution of this residue by alanine resulted in loss of activity. The E349G variant retained 5%activity for the coupled reaction, suggesting that coordinating 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 Ile267 Ala268 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 coordination 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 coordination position.
Original languageEnglish
Pages (from-to)1179-1189
Number of pages11
JournalBiochemical Journal
Issue number9
Publication statusPublished - 1 May 2016


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