Structural and Functional Basis for (S)-Allantoin Formation in the Ureide Pathway

Kwangsoo Kim,Jinseo Park,Sangkee Rhee

doi:10.1074/jbc.m703211200

Abstract

The ureide pathway, which mediates the oxidative degradation of uric acid to (S)-allantoin, represents the late stage of purine catabolism in most organisms. The details of uric acid metabolism remained elusive until the complete pathway involving three enzymes was recently identified and characterized. However, the molecular details of the exclusive production of one enantiomer of allantoin in this pathway are still undefined. Here we report the crystal structure of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase, which catalyzes the last reaction of the pathway, in a complex with the product, (S)-allantoin, at 2.5-A resolution. The homodimeric helical protein represents a novel structural motif and reveals that the active site in each monomer contains no cofactors, distinguishing this enzyme mechanistically from other cofactor-dependent decarboxylases. On the basis of structural analysis, along with site-directed mutagenesis, a mechanism for the enzyme is proposed in which a decarboxylation reaction occurs directly, and the invariant histidine residue in the OHCU decarboxylase family plays an essential role in producing (S)-allantoin through a proton transfer from the hydroxyl group at C4 to C5 at the re-face of OHCU. These results provide molecular details that address a longstanding question of how living organisms selectively produce (S)-allantoin.

Highlights

AUGUST 10, 2007 VOLUME 282 NUMBER 32 further enzyme-dependent degradation
Overall Structure of OHCU Decarboxylase—The OHCU decarboxylase of A. thaliana consists of the N-terminal 161 residues of a bifunctional enzyme fused with HIU hydrolase [8, 17] (Fig. 1)
We have presented structural and functional evidence that OHCU decarboxylase, an enzyme of the ureide pathway, contains a novel helical structural fold and is a cofactor-independent enzyme

Summary

EXPERIMENTAL PROCEDURES

Protein Purification and Crystallization—For the expression of His-tagged recombinant A. thaliana OHCU decarboxylase, a portion of the cDNA (483 bp; residues 1–161) encoding the A. Crystal Structure of OHCU Decarboxylase thaliana bifunctional enzyme was amplified using two sequence-specific primers (Table 1). The proteins were expressed as N-terminal His-tagged proteins and purified as described above for the wild-type and mutant OHCU decarboxylases and as described previously for HIU hydrolase [14]. The purified enzyme was concentrated to ϳ1.0 mg/ml in 50 mM Tris-HCl (pH 8.0) for the wild-type and mutant OHCU decarboxylases, and ϳ1.0 mg/ml in 20 mM TrisHCl (pH 8.0) and 200 mM NaCl for HIU hydrolase. Uric acid (50 ␮M) in 1 ml of 50 mM potassium phosphate buffer, pH 7.6, was incubated in the cuvette with urate oxidase (1 unit) and HIU hydrolase (4.0 ␮g), and once OHCU reached its maximal concentration, OHCU decarboxylase (4.0 ␮g) was added. The formation of allantoin in the presence of the three enzymes of the ureide pathway was measured in the range of 200 –360 nm at specified incubation times

RESULTS

Data collection

DISCUSSION