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Comparative Study
. 2004 Apr;186(7):1945-58.
doi: 10.1128/JB.186.7.1945-1958.2004.

Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases

Matthew W Vetting  1 , Lawrence P Wackett, Lawrence Que Jr, John D Lipscomb, Douglas H Ohlendorf
Affiliations
Comparative Study

Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases

Matthew W Vetting et al. J Bacteriol. 2004 Apr.

Abstract

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.

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Figures

FIG. 1.
FIG. 1.
Divergent stereo of a Cα trace of a monomer of Bf 2,3-HPCD. MndD and Bf 2,3-HPCD monomers are composed of an N-terminal domain (red), a C-terminal domain (blue), and a lid domain (cyan). The substrate binds to the iron (green sphere) in a pocket in the barrel of the C-terminal domain. Every 20th residue is labeled. Residues involved in the secondary structural elements are listed at the bottom.
FIG. 2.
FIG. 2.
2Fo-Fc electron density for the ligands to the iron in the native and complex structures of Bf 2,3-HPCD and MndD. Electron density is contoured at 1σ. (A) Bf 2,3-HPCD with no substrate (Native 2, I41); (B) Bf 2,3-HPCD soaked with 1 mM HPCA; (C) MndD with no substrate (Native 2, C2); (D) MndD soaked with 1 mM HPCA. This figure was produced by use of the modeling program O (37).
FIG. 3.
FIG. 3.
Divergent stereo diagram of the interaction of the substrate with residues of the active site in MndD. Important potential hydrogen bonds are shown as dotted lines. The active-site Mn2+ is shown as a light gray sphere, while a single equatorial solvent is shown as a dark sphere. This figure was prepared by use of SETOR (23).
FIG. 4.
FIG. 4.
Electron density and residue interactions in the region surrounding the binding site of the acetate group of the substrate in the P3221 crystal form of Bf 2,3-HPCD. For purposes of clarity, ligands to the iron and their respective densities are not shown. Additional electron density at the iron is modeled by two solvent molecules. Dotted lines, potential hydrogen bonds. This figure was produced by use of the modeling program O (37).
FIG. 5.
FIG. 5.
Divergent stereo diagram of the superposition of MndD (green) and Bf 2,3-HPCD (red) in an area around the active site. Sequence differences for Bf 2,3-HPCD are given after the sequence number. Significant differences occur only 10 to 15 Å away from the iron. This figure was prepared by use of SETOR.
FIG. 6.
FIG. 6.
Proposed enzyme mechanism. The typical geometry (E267OE1, H214NE1, O2, and HPCAO4 equatorial ligands) is rotated (H155NE1, H214NE1, HPCAO3, and HPCAO4 equatorial ligands) for clarity and for comparison to previously published mechanism diagrams (63, 71).
FIG. 7.
FIG. 7.
Comparison of the binding characteristics of intradiol and extradiol dioxygenases. Numbering for extradiol enzymes is based on that of Bf 2,3-HPCD; numbering for intradiol dioxygenases is based on that of protocatechuate 3,4-dioxygenase from P. putida. Asterisks indicate the position at which oxygen is proposed to attack during the formation of the peroxy intermediate. The proposed location of oxygen is indicated.
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