Steve Ealick's Research Group

Ralstonia metallidurans 3-Hydroxyanthranilate-3,4-dioxygenase

PDB files:

1YFU unliganded HAD

1YFW HAD complexed with 4-chloro-3-hydroxyanthranilic acid and O2

1YFX HAD complexed with 4-chloro-3-hydroxyanthranilic acid and NO

1YFY HAD complexed with 3-hydroxyanthranilic acid


3-Hydroxyanthranilate-3,4-dioxygenase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate (HAA) to yield 2-amino-3-carboxymuconic semialdehyde. The reaction is the final enzymatic step in the tryptophan to quinolinate biosynthetic pathway, which is of substantial interest because quinolinate is the precursor to the pyridine ring of the nicotinamide cofactors. It had been thought that the biosynthesis of quinolinate from tryptophan was unique to eukaryotes, while in prokaryotes quinolinate is usually derived from aspartate and dihydroxyacetone phosphate. The identification and overexpression of a bacterial HAD from R. metallidurans greatly facilitated our structural and mechanistic studies.

The core motif of the monomer structure is a jellyroll β-barrel formed by two anti-parallel β-sheets with a topology that is characteristic of a cupin barrel fold. Each monomer contains two iron binding sites. The catalytic iron is buried deep inside the β-barrel The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by cysteine residues. The two iron sites are separated by about 24 Å.

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The overall structure of HAD is a homodimer with with a subunit topology that is characteristic of the cupin barrel fold. The approximate dimensions are 80 Å x 40 Å x30 Å and the two monomers are related by a crystallographic twofold axis. The arrangement of the two cupin barrels in a dimer resembles bicupin structures in which each monomer has two cupin domains, such as the bicupin monomer unit in oxalate decarboxylase.


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A complete active site, which contains an Fe(III) ion, is found in each HAD monomer and is buried deeply inside the cupin barrel. This figure shows 4-chloro-3-hydroxyanthranilic acid bound to the catalytic iron at the active site.


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Zhang Y, Colabroy KL, Begley TP, and Ealick SE. Structural studies on 3-hydroxyanthranilate-3,4-dioxygenase: the catalytic mechanism of a complex oxidation involved in NAD biosynthesis. Biochemistry 44:7632-7643 (2005).

Colabroy KL, Zhai H, Li T, Ying Ge1, Zhang Y, Liu A, Ealick SE, McLafferty FW and Begley TP, The mechanism of inactivation of 3-hydroxyanthranilate-3,4-dioxygenase by 4-chloro-3-hydroxyanthranilate. Biochemistry 44:7623-7631 (2005).


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