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The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase - PubMed

Comparative Study

The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase

Iris M Fritze et al. Plant Physiol. 2004 Apr.

Abstract

The transformation of 4-hydroxyphenylpyruvate to homogentisate, catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPPD), plays an important role in degrading aromatic amino acids. As the reaction product homogentisate serves as aromatic precursor for prenylquinone synthesis in plants, the enzyme is an interesting target for herbicides. In this study we report the first x-ray structures of the plant HPPDs of Zea mays and Arabidopsis in their substrate-free form at 2.0 A and 3.0 A resolution, respectively. Previous biochemical characterizations have demonstrated that eukaryotic enzymes behave as homodimers in contrast to prokaryotic HPPDs, which are homotetramers. Plant and bacterial enzymes share the overall fold but use orthogonal surfaces for oligomerization. In addition, comparison of both structures provides direct evidence that the C-terminal helix gates substrate access to the active site around a nonheme ferrous iron center. In the Z. mays HPPD structure this helix packs into the active site, sequestering it completely from the solvent. In contrast, in the Arabidopsis structure this helix tilted by about 60 degrees into the solvent and leaves the active site fully accessible. By elucidating the structure of plant HPPD enzymes we aim to provide a structural basis for the development of new herbicides.

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Figures

Figure 1.
Figure 1.

a, Reaction catalyzed by HPPD. b, Structures of HPPD inhibitors sulcotrione (a), mesotrione (b) and NTBC (c), which are based on a triketone backbone and may mimic an HPPD reaction coordinate transition state or intermediate.

Figure 2.
Figure 2.

Structure of HPPD. a, Ribbon representation of the Z. mays HPPD dimer. The view is down the 2-fold axis. b, The Z. mays HPPD monomer shows two open β-barrel domains (green and blue), which are both built up by two similar modules (light and dark colors). The catalytic iron atom in the C-terminal domain is shown as a red sphere. c, Superimposition of HPPD monomers of Z. mays (blue), Arabidopsis (green), and P. fluorescens (light brown).

Figure 3.
Figure 3.

The alignment of different plant HPPD sequences is structure based on the sequences of Z. mays, Arabidopsis, and P. fluorescens. Numbering is given for Z. mays HPPD according to Maxwell et al., (1997). Secondary structures are color coded like in Figure 2 and were numbered according to Serre et al. (1999). The secondary structure assignment of P. fluorescens HPPD is depicted in light gray. Black lines indicate loop regions, whereas disordered residues of Z. mays HPPD are indicated without the corresponding lines. Structural elements mediating dimerization are marked by a black line above the Z. mays sequence with residues directly involved in dimer contacts shown in green. Amino acids highlighted in blue are invariant, conservative exchanges are depicted in light-green. The iron coordinating residues are marked by red triangles. Active site residues are boxed. The residues building the rigid cavity of the articular are marked by a black triangle.

Figure 4.
Figure 4.

Quaternary structure of bacterial and plant HPPDs. The Z. mays HPPD dimer (blue) uses an orthogonal molecular surface compared to the dimer contacts present in the P. fluorescens tetramer (brown to orange) of 222 point symmetry.

Figure 5.
Figure 5.

Gating to the active site. Z. mays HPPD is shown in blue, Arabidopsis in green, and P. fluorescens in light brown. The highly conserved Asn-416 forms the hinge region around which the C-terminal α-helix H11 rotates to open the active site, whereas Leu-361 and Asp-363 function as rigid articular cavity. The region around α-helix H8 remains rigid, whereas the loop connecting β-strands B3 and C3 is flexible and presumably adopts to the movement of the gating α-helix H11.

Figure 6.
Figure 6.

Active site architecture. a, Stereo picture showing the 2Fo-Fc electron density at the 1σ level at the active site of Z. mays HPPD. The active site iron shows an octahedral coordination sphere of three amino acid ligands and three water molecules. b, Superimposition of the active site structures of Z. mays (blue), Arabidopsis (green), and P. fluorescens (light brown) HPPD. The view includes residues approximately 14 Å around the catalytically active iron atom. Ile-220, Leu-274, Met-302, Leu-304, Ile-373, Leu-386, Ile-388, and Lys-414 in the front were omitted for clarity. The C-terminal gating helices are shown as coils.

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