Publications

• Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes.

  Ramazzina I., Folli C., Secchi A., Berni R., Percudani R.

  Mammals that degrade uric acid are not affected by gout or urate kidney stones. It is not fully understood how they convert uric acid into the much more soluble allantoin. Until recently, it had long been thought that urate oxidase was the only enzyme responsible for this conversion. However, deta ... >> More

  Mammals that degrade uric acid are not affected by gout or urate kidney stones. It is not fully understood how they convert uric acid into the much more soluble allantoin. Until recently, it had long been thought that urate oxidase was the only enzyme responsible for this conversion. However, detailed studies of the mechanism and regiochemistry of urate oxidation have called this assumption into question, suggesting the existence of other distinct enzymatic activities. Through phylogenetic genome comparison, we identify here two genes that share with urate oxidase a common history of loss or gain events. We show that the two proteins encoded by mouse genes catalyze two consecutive steps following urate oxidation to 5-hydroxyisourate (HIU): hydrolysis of HIU to give 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and decarboxylation of OHCU to give S-(+)-allantoin. Urate oxidation produces racemic allantoin on a time scale of hours, whereas the full enzymatic complement produces dextrorotatory allantoin on a time scale of seconds. The use of these enzymes in association with urate oxidase could improve the therapy of hyperuricemia. << Less

  Nat. Chem. Biol. 2:144-148(2006) [PubMed] [EuropePMC]

• The structure of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase provides insights into the mechanism of uric acid degradation.

  Cendron L., Berni R., Folli C., Ramazzina I., Percudani R., Zanotti G.

  The complete degradation of uric acid to (S)-allantoin, as recently elucidated, involves three enzymatic reactions. Inactivation by pseudogenization of the genes of the pathway occurred during hominoid evolution, resulting in a high concentration of urate in the blood and susceptibility to gout. H ... >> More

  The complete degradation of uric acid to (S)-allantoin, as recently elucidated, involves three enzymatic reactions. Inactivation by pseudogenization of the genes of the pathway occurred during hominoid evolution, resulting in a high concentration of urate in the blood and susceptibility to gout. Here, we describe the 1.8A resolution crystal structure of the homodimeric 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, which catalyzes the last step in the urate degradation pathway, for both ligand-free enzyme and enzyme in complex with the substrate analogs (R)-allantoin and guanine. Each monomer comprises ten alpha-helices, grouped into two domains and assembled in a novel fold. The structure and the mutational analysis of the active site have allowed us to identify some residues that are essential for catalysis, among which His-67 and Glu-87 appear to play a particularly significant role. Glu-87 may facilitate the exit of the carboxylate group because of electrostatic repulsion that destabilizes the ground state of the substrate, whereas His-67 is likely to be involved in a protonation step leading to the stereoselective formation of the (S)-allantoin enantiomer as reaction product. The structural and functional characterization of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase can provide useful information in view of the potential use of this enzyme in the enzymatic therapy of gout. << Less

  J. Biol. Chem. 282:18182-18189(2007) [PubMed] [EuropePMC]

• Structural and mechanistic studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase.

  French J.B., Ealick S.E.

  The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here w ... >> More

  The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K(i) of 30 ± 2 μM. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation. << Less

  J Biol Chem 285:35446-35454(2010) [PubMed] [EuropePMC]

• Structural and functional basis for (S)-allantoin formation in the ureide pathway.

  Kim K., Park J., Rhee S.

  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 characte ... >> More

  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. << Less

  J. Biol. Chem. 282:23457-23464(2007) [PubMed] [EuropePMC]