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

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-Å 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 cofactordependent 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.The purine degradation pathway has an essential role in nitrogen metabolism in most organisms. In this catabolism pathway, inosine monophosphate, which is the final product of de novo purine biosynthesis, is degraded through sequential enzymatic steps into uric acid, which contains a high level of nitrogen (1). Most organisms, including some bacteria, plants, and animals, utilize a common pathway for uric acid metabolism and produce stereospecific (S)-allantoin as the final product (2, 3). Subsequently, (S)-allantoin, which contains an even ratio of nitrogen to carbon, is used as a nitrogen source through further enzyme-dependent degradation. This metabolism of uric acid, a process named the ureide pathway, has a pivotal role in transforming the nitrogen that is fixed in leguminous plants (2, 3) and also plays a crucial role in some bacteria under nitrogen-limited conditions (4). This pathway was once thought to be executed by a single enzyme, urate oxidase (5), but recent investigations have revealed two additional enzymes in the pathway (Scheme 1). The pathway is initiated by urate oxidase, producing the unstable 5-hydroxyisourate (HIU), 3 which has a half-life of ϳ30 min in aqueous solution, followed by hydrolysis by HIU hydrolase to produce OHCU, which also undergoes spontaneous degradation (6 -8). The third enzyme, OHCU decarboxylase, was identified through the phylogenetic analysis of a whole genome and has been shown to catalyze the decarboxylation of OHCU, producing the stereospecific (S)-allantoin (8). These observations are consistent with the previous identification of two chemically distinct labile intermediates produced by urate oxid...

Conserved Alternative Splicing of Arabidopsis Transthyretin-Like Determines Protein Localization and S-Allantoin Synthesis in Peroxisomes

“…On the other hand, the TTL protein contains a sequence resembling a peroxisome targeting signal (Hennebry et al, 2006), and it has been found as a component of Arabidopsis leaf peroxisomes (Reumann et al, 2007). Moreover, structural studies have shown that the Urad domain of TTL binds S-allantoin and has structural similarity to vertebrate OHCU decarboxylase (Kim et al, 2007). Altogether, these observations suggest that the Arabidopsis TTL protein plays a dual role in plant growth, both in modulating brassinosteroid hormone responses and ureide synthesis.…”

Structural and Functional Insights into (S)-Ureidoglycine Aminohydrolase, Key Enzyme of Purine Catabolism in Arabidopsis thaliana

“…The modifications were performed using the QuikChange method (Agilent) to introduce a silent mutation at the sequences for NdeI and XhoI sites residing in the internal region of the AtUGlyAH gene. The N-terminal truncated variants of AtUGlyAH, from which the signal peptide (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and an additional N-terminal region had been removed, were amplified using a pair of sequence-specific primers (supplemental Table S1). The resulting PCR products were introduced into the NdeI and XhoI sites of a modified pET-28a vector (Merck) containing a tobacco etch virus protease cleavage site at the junction between a His 5 tag and a multiple cloning site.…”

Emergent Decarboxylase Activity and Attenuation of α/β-Hydrolase Activity during the Evolution of Methylketone Biosynthesis in Tomato

“…During the decarboxylative cleavage of the C1-C2 bond, a negative charge ultimately develops in the form of a C2 carbanion. Many decarboxlases employ cofactors to stabilize carbanion formation (Begley and Ealick, 2004), although cofactorindependent decarboxylases have been recently discovered (Appleby et al, 2000;Harris et al, 2000;Miller et al, 2000;Wu et al, 2000;Cendron et al, 2007;Kim et al, 2007;Chan et al, 2009). 3-Keto acids carry an inherent electron sink in the 3-carbonyl group, a property underlying their implied instability (Jencks, 1969).…”