Structures of aminophenol dioxygenase in complex with intermediate, product and inhibitor

Abstract

Dioxygen activation by nonhaem Fe(II) enzymes containing the 2-His-1-carboxylate facial triad has been extensively studied in recent years. Here, crystal structures of 2-aminophenol 1,6-dioxygenase, an enzyme that represents a minor group of extradiol dioxygenases and that catalyses the ring opening of 2-aminophenol, in complex with the lactone intermediate (4Z,6Z)-3-iminooxepin-2(3H)-one and the product 2-aminomuconic 6-semialdehyde and in complex with the suicide inhibitor 4-nitrocatechol are reported. The Fe-ligand binding schemes observed in these structures revealed some common geometrical characteristics that are shared by the published structures of extradiol dioxygenases, suggesting that enzymes that catalyse the oxidation of noncatecholic compounds are very likely to utilize a similar strategy for dioxygen activation and the fission of aromatic rings as the canonical mechanism. The Fe-ligation arrangement, however, is strikingly enantiomeric to that of all other 2-His-1-carboxylate enzymes apart from protocatechuate 4,5-dioxygenase. This structural variance leads to the generation of an uncommon O(-)-Fe(2+)-O(-) species prior to O(2) binding, which probably forms the structural basis on which APD distinguishes its specific substrate and inhibitor, which share an analogous molecular structure.

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

“…This oxygenated intermediate also inherits the monodentate coordination status found in the previous lactone intermediate. These observations are consistent with findings in other extradiol dioxygenases that the newly formed carboxyl group binds to the iron in a monodentate fashion with the products retaining a more cyclized conformation (29,45,46).…”

“…A denticity change, from one to two, is observed in the product bound complex. Most of the products observed through in crystallo reaction in extradiol dioxygenases maintain a cyclized conformation (29,45,46), as seen in (E•P) I of HAO. A linearized product like (E•P) II is less commonly seen bound at the active site in dioxygenases in the degradation of aromatic molecules (40).…”

Oxidative opening of the aromatic ring: Tracing the natural history of a large superfamily of dioxygenase domains and their relatives

“…Prior experiments have shown that some members of the classical LigB family function as homodimers (24,28). A range of other configurations have also been described: the gallate dioxygenase GalA, a member of the classical LigB family, functions as a homotrimer (29); the APD family forms a heterodimer between active and inactive paralogs (30); and the YgiD-like family has thus far been characterized as a functional monomer in bacteria (31) and plants (32). The significance of potential multimerization in the Memo clade remains unexplored.…”

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover

“…CtAPD is an extradiol ring-cleavage dioxygenase whose X-ray structure is available and its sequence identity to DHPAOs in Cluster 1 is ~21–25%. Based on the crystal structure of CtAPD (PDB codes: 3VSH and 3VHI), the active-site residues His13, His62, and Glu251 are ligated with a mononuclear non-heme Fe(II) center and Tyr129 and His195 are involved in formation of a second sphere metal coordination ( S1 Fig ) [ 42 ]. Based on the sequence analysis ( S1 Fig ), the three active-site residues involved in Fe(II) coordination of CtAPD are all conserved in PaDHPAO as well as other DHPAOs in Cluster 1 (His12, His57, and Glu239 of PaDHPAO as highlighted in yellow in S1 Fig ).…”

“…The other two active-site residues (highlighted in cyan, Tyr129 and His195) are also conserved in PaDHPAO (Tyr125 and His186) and other DHPAOs in Cluster 1. These two residues are important for second sphere metal-coordination and were shown to be important for catalysis [ 42 ]. (B) The active site structure of CtAPD co-crystallized with 4-nitrocatechol (4NC) shows the involvement of all these conserved residues in metal coordination and substrate binding.…”