Catalysis, Stereochemistry, and Inhibition of Ureidoglycolate Lyase

Abstract

Ureidoglycolate lyase (UGL, EC 4.3.2.3) catalyzes the breakdown of ureidoglycolate to glyoxylate and urea, which is the final step in the catabolic pathway leading from purines to urea. Although the sequence of enzymatic steps was worked out nearly 40 years ago, the stereochemistry of the uric acid degradation pathway and the catalytic properties of UGL have remained very poorly described. We now report the first direct investigation of the absolute stereochemistry of UGL catalysis. Using chiral chromatographic analyses with substrate enantiomers, we demonstrate that UGL catalysis is stereospecific for substrates with the (S)-hydroxyglycine configuration. The first potent competitive inhibitors for UGL are reported here. These inhibitors are compounds which contain a 2,4-dioxocarboxylate moiety, designed to mimic transient species produced during lyase catalysis. The most potent inhibitor, 2,4-dioxo-4-phenylbutanoic acid, exhibits a K I value of 2.2 nM and is therefore among the most potent competitive inhibitors ever reported for a lyase enzyme. New synthetic alternate substrates for UGL, which are acyl-␣-hydroxyglycine compounds, are described. Based on these alternate substrates, we introduce the first assay method for monitoring UGL activity directly. Finally, we report the first putative primary nucleotide and derived peptide sequence for UGL. This sequence exhibits a high level of similarity to the fumarylacetoacetate hydrolase family of proteins. Close mechanistic similarities can be visualized between the chemistries of ureidoglycolate lyase and fumarylacetoacetate hydrolase catalysis.Ureidoglycolate lyase (UGL, 1 EC 4.3.2.3) and allantoicase (Acase, EC 3.5.3.4) are the enzymes that catalyze the final steps in the catabolic pathway leading from purines to urea. In this pathway ( Fig. 1), allantoic acid is formed from allantoin by allantoinase, Acase then converts allantoic acid to ureidoglycolate and urea, and, finally, UGL catalyzes the breakdown of ureidoglycolate to glyoxylate and urea. Acase was first found independently in frog liver by Krebs and Weil (1) and in the mycelium of Aspergillus niger by Brunel (2). UGL was first described by Valentine et al. (3,4) in Streptococcus allantoicus and in a strain of Pseudomonas. It has since been established that UGL and Acase are both functional in many bacteria, yeast, fish, and rat (5-16), whereas in some species only one of these activities has been detected (2, 17-23).The stereochemistry of UGL catalysis, and indeed of the uric acid degradation pathway as a whole, has remained poorly described for many years. Conflicting results have been reported as to whether UGL reacts preferentially with (Ϫ)-ureidoglycolate (5, 6, 9, 10, 18) or with (ϩ)-ureidoglycolate (17). Moreover, allantoinase reacts with both enantiomers of allantoin, and Acase has been reported to produce ureidoglycolate exhibiting a negative rotation, but, paradoxically, to also convert ureidoglycolate exhibiting a positive rotation to glyoxylate and urea (11). Thus, the stereospecificity of U...

Role of urea in microbial metabolism in aquatic systems: a biochemical and molecular review

“…The rate of transport of urea into the cell is measured as urea uptake activity. Urea is also produced internally as a byproduct of the urea cycle, amino acid catabolism (Mobley & Hausinger 1989, Antia et al 1991, Siewe et al 1998, Beckers et al 2004, and purine catabolism (Vogels & Van der Drift 1976, McIninch et al 2003, Allen et al 2006. Urea produced from catabolism/salvage pathways can be either excreted into the environment (Bronk et al 1998, Jørgensen et al 1999b, Berg & Jørgensen 2006 or further hydrolyzed to NH 4 + and CO 2 by the enzyme urease (encoded by the ure genes; Mobley & Hausinger 1989, Antia et al 1991, Zehr & Ward 2002 or by ATP: urea amidolyase (UALase, encoded by DUR1,2; Antia et al 1991, Hausinger 2004.…”

Role for an Essential Tyrosine in Peptide Amidation

“…UGL was first described in Streptococcus allantoicus and Pseudomonas (19,20). UGL activity has been studied in bacteria, yeast, fish, and rat, and UGL has been purified from fish liver (21), chick pea (22), and Burkholderia cepacia (23). The only sequence data available are for B. cepacia UGL and place it in the fumarylacetoacetate hydrolase family (23).…”

“…UGL activity has been studied in bacteria, yeast, fish, and rat, and UGL has been purified from fish liver (21), chick pea (22), and Burkholderia cepacia (23). The only sequence data available are for B. cepacia UGL and place it in the fumarylacetoacetate hydrolase family (23). Fumarylacetoacetate hydrolase (FAH; EC 3.7.1.2) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, yielding fumarate and acetoacetate in the final step of Phe and Tyr degradation (24).…”

Crystal Structure of Yeast Allantoicase Reveals a Repeated Jelly Roll Motif

“…Ureidoglycolate lyases are specialized enzymes that catalyze the conversion of ureidoglycolate into urea and glyoxylate, but no individual gene associated with this activity has been reported in yeast. The mechanism and partial sequence of the ureidoglycolate lyase of Burkholderia cepacia were recently characterized, but this did not reveal any yeast orthologues (14). Apart from the pocket described above, no second conserved surface patch is present in yeast allantoicase, and both reactions probably take place at the same active site.…”

“…Almost nothing is known on the reaction mechanism or structure of allantoicases, and mechanistic information on any hydrolase acting on linear amidines is scarce (14). We here present the 2.6-Å crystal structure of the allantoicase from Saccharomyces cerevisiae, which is the first structure for this enzyme family.…”