Identification and Purification of Hydroxyisourate Hydrolase, a Novel Ureide-metabolizing Enzyme*

We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules ∼100-fold to apparent homogeneity with a final specific activity of 10 μmol/min/mg. The enzyme exhibited a native molecular mass of ∼68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 ± 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and theK m for 5-hydroxyisourate was determined to be 15 μm. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.

Nitrogen is a key component of plant metabolism, and its availability often limits the growth of important crop plants. Leguminous plants are able to acquire their nitrogen through association with bacterial symbionts in the root nodules, which fix atmospheric nitrogen to form NH 4 ϩ through the action of nitrogenase. The fixed nitrogen is then transported from the bacteria into the host cell cytoplasm, where it is assimilated into organic form and used for the synthesis of nucleic acids, amino acids, and secondary products.
The ureides are the major form of nitrogen transport molecules in tropical legumes such as soybean. In nodulated soybean ureides constitute 70 -80% of the organic nitrogen in the xylem sap (1). The ureides are efficient nitrogen transport species; the ratio of C to N in allantoin and allantoate is 1:1, so minimal carbon is diverted from other metabolic functions in support of nitrogen transport. The conversion of inorganic nitrogen into organic forms is an energetically expensive process, however. It has been estimated that 68 ATPs are required for the synthesis of allantoin if the cost of N 2 fixation is included (2). Thus, one would expect mechanisms for the efficient utilization of fixed nitrogen to have arisen.
Purine oxidation is the major route for ureide biogenesis, and the so-called ureide pathway is constituted by the enzymes that carry out the conversion of IMP to allantoin and allantoate. It is commonly considered that the role of urate oxidase in this pathway is the conversion of urate to allantoin (3). However, it has recently been demonstrated that allantoin is not the true product of the urate oxidase reaction (4,5). Urate oxidase catalyzes the conversion of urate to 5-hydroxyisourate, which decomposes cleanly to allantoin under most in vitro conditions.
The half-life of HIU 1 at neutral pH is on the order of 30 min in vitro (6). Because the flux through the ureide pathway is critical for nitrogen fixation and metabolism, it is difficult to conceive that the nonenzymatic conversion of HIU to allantoin is the mechanism for ureide synthesis in vivo. A second confounding factor arises from the fact that nonenzymatic decomposition of HIU generates racemic allantoin (7). However, soybean allantoinase is specific for (S)-allantoin (8), and we are not aware of reports of an allantoin racemase occurring in plants. The half-time for racemization of allantoin at neutral pH is ϳ10 h, 2 so again, it would seem that nonenzymatic chemistry is far too slow to support the ureide pathway. These considerations raise the possibility that there exist previously unrecognized enzymes which are responsible for the stereospecific conversion of HIU to (S)-allantoin.
In this report we present evidence for the existence of a novel enzyme which catalyzes the hydrolysis of HIU to form OHCU. OHCU has previously been characterized as the next species in the pathway leading from HIU to allantoin under nonenzymatic conditions (Ref. 9 and Scheme 1). We have purified the enzyme, which we designate hydroxyisourate hydrolase, to apparent homogeneity. Sequence data obtained at the amino terminus confirm the novelty of this protein.

MATERIALS AND METHODS
Uric acid and catalase were obtained from Sigma. Recombinant soybean urate oxidase was purified as described (4). Protein sequencing was performed at the University of Missouri Protein Core Facility.
Substrate Preparation-5-Hydroxyisourate was generated by urate oxidase-catalyzed turnover of urate. Approximately 1.5 units of recombinant urate oxidase was added to a 3.0 mM urate solution in 50 mM potassium phosphate buffer, pH 7.2, and the solution was gently bubbled with O 2 . The reaction was monitored spectrophotometrically, and when HIU formation was maximal the enzyme was removed by ultrafiltration, and the filtrate was stored as aliquots at Ϫ80°C. The HIU concentration was determined spectrophotometrically (⑀ 302 ϭ 8300 M/cm; Ref. 6). We estimate that the HIU solutions contained no more than 10% residual urate.
Enzyme Assays-HIUHase activity was routinely measured by monitoring the disappearance of HIU at 302 nm. The standard assay mix contained 100 mM potassium phosphate buffer, pH 7.2, and 100 M HIU; the reaction was initiated by addition of enzyme. For each assay a control reaction from which enzyme was omitted was also monitored to determine the rate of nonenzymatic HIU decomposition. The background rate, which typically did not exceed 25% of the enzyme-cata-* This work was supported by United States Department of Agriculture Grant 98-35305-6548. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Biochemistry, University of Missouri, 117 Schweitzer Hall, Columbia, MO 65211. Tel: 573-882-7968; Fax: 573-884-4812; E-mail: tiptonp@missouri.edu. lyzed rate, was subtracted from each enzyme activity measurement to determine the actual HIUHase activity. One unit of activity was defined to be that amount of enzyme that turned over 1 mol of HIU/min under the above conditions at 25°C.
HIUHase activity was also detected in crude soybean root nodule extract by monitoring the change in optical activity of a solution containing HIU generated in situ by purified urate oxidase. Reactions were conducted in 1-mm pathlength cuvettes, and circular dichroism spectra were obtained with an Aviv 62DS spectropolarimeter.
Enzyme Purification-The purification of HIUHase was accomplished from soybean root nodule extracts (10) generously supplied by Prof. David Emerich (Department of Biochemistry, University of Missouri). All enzyme purification steps were carried out at 4°C. Nucleic acids were removed from the crude extract by the addition of protamine sulfate to a final concentration of 1% and subsequent centrifugation. Solid ammonium sulfate was added to the supernatant to reach 65% saturation, and the pelleted protein obtained after centrifugation was dissolved in a minimal amount of 50 mM potassium phosphate, pH 7.3, containing 1 mM phenylmethylsulfonyl fluoride and 2 M NaCl. The solution was applied at a flow rate of 2 ml/min to a phenyl-Sepharose column (1.2 ϫ 20 cm) and developed with a step gradient of decreasing NaCl concentration. HIUHase was eluted with buffer containing no salt. Active fractions were concentrated by addition of ammonium sulfate to a final concentration of 65% saturation. The pellet obtained after centrifugation was dissolved in 50 mM Tris-HCl, pH 7.8, containing 50 mM NaCl and 10% glycerol (v/v) and applied to a gel filtration column (Superdex-200, 1.2 ϫ 100 cm). The column was developed with a flow rate of 0.5 ml/min. Fractions containing the highest activity were combined and applied to an immobilized metal affinity column (1 ϫ 10 cm, Chelate Spherilose; Isco). The resin was charged with copper according to the manufacturer's instructions. After sample application, the column was washed with 3-4 column volumes of 20 mM Tris-HCl, pH 7.8, containing 0.5 M NaCl, 1 mM imidazole, and 5% glycerol (v/v). The enzyme was eluted with the same buffer containing 20 mM imidazole, and active fractions were concentrated by ultrafiltration. The sample was exchanged into 20 mM potassium phosphate, pH 6.8, by repeated dilution and concentration via ultrafiltration and applied to a hydroxy-apatite column (1 ϫ 5 cm). After initially washing the column with 3 column volumes of 20 mM potassium phosphate, pH 6.8, a linear gradient to 200 mM potassium phosphate, pH 6.8, over 50 ml was formed to elute the enzyme.
Product Identification-Approximately 8 g of HIUHase and 1 unit of purified urate oxidase were added to a 1-ml solution containing 2.5 mM [4,6-13 C 2 ]urate (9) and 400 units of catalase in 95 mM sodium phosphate in D 2 O, pD 7. The solution was transferred to a 5-mm NMR tube, and 13 C NMR spectra were obtained at regular time intervals at 17°C. The 13 C NMR spectra were acquired with a Bruker AVANCE DRX500 spectrometer using a sweep width of 49,682 Hz. 1,4-Dioxane was included as a chemical shift standard.

RESULTS AND DISCUSSION
Detection of HIUHase-The observation that urate oxidase catalyzed the conversion of urate, not to allantoin, but to 5-hydroxyisourate prompted us to search for an enzyme that utilized 5-hydroxyisourate as a substrate. The effect of added crude soybean root nodule extract to a solution of HIU generated in situ is shown in Fig. 1. It is clear that the extract contained a component that caused the rapid disappearance of HIU. Boiled root nodule extract had no effect on the kinetics of  ppm, respectively. The signal from C4 is not apparent in OHCU, because it equilibrates with its hydrate. Note that the 13 C signals in allantoin carboxylate are doublets because of the coupling between the adjacent 13 C atoms. The signals at ϳ62 and 72 ppm arise from glycerol in the enzyme solutions, which were added to the reaction; their intensities do not change during the course of the experiment. SCHEME 1 decomposition of HIU. Monitoring the reaction by absorbance spectroscopy confirmed that the disappearance of the optically active HIU was caused by conversion to another species, not racemization.
Purification of HIUHase-HIUHase was purified from extracts of 4-week-old soybean root nodules using standard protein purification techniques. Table I shows the results from a typical purification of HIUHase. Because of severe interference by polyphenolic compounds during the measurements of activity and protein content in the crude extract and protamine sulfate fractions, the ammonium sulfate fraction is designated as the initial fraction for the purpose of calculating the purity and recovery of activity at each purification step.
The mass of the enzyme under nondenaturing conditions was determined by gel filtration chromatography on a calibrated Superdex-200 column; HIUHase eluted with an apparent molecular mass of 68 kDa. SDS-polyacrylamide gel electrophoresis of the purified protein yielded a single band upon staining with Coomassie Blue, which had an apparent molecular mass of 68 Ϯ 2 kDa (Fig. 2). Thus, HIUHase appears to be a monomer.
The purified protein was subjected to automated Edman degradation, and the sequence of the first 19 amino acids at the amino terminus was determined to be ADNYSRDDFPLD-FVFVFGS. Searches of available data bases did not reveal any proteins with significant homology to this peptide sequence.
Identification of the Reaction Product-The product of the HIUHase reaction was identified by 13 C NMR spectroscopy. The 13 C NMR spectra of HIU and the intermediates on the pathway to allantoin formation (Scheme 1) have previously been assigned (9). As shown in Fig. 3, in the presence of urate oxidase and HIUHase, [4,6-13 C 2 ]urate is rapidly converted to HIU and OHCU, and the subsequent conversion of OHCU to allantoin is evident. In experiments conducted under very similar conditions but in the absence of HIUHase, the formation of HIU before the appearance of OHCU is apparent, and the complete conversion of HIU to allantoin requires several hours (9).
Kinetic Properties-HIUHase obeyed normal Michaelis-Menten kinetics; the K m for HIU was determined to be 15 Ϯ 3 M, and the turnover number was 11/s. Because we were concerned that residual urate in our solutions of HIU might effect the kinetics of the reaction, we examined the effect of urate on the activity of HIUHase. Urate did not inhibit HIUHase up to concentrations of 1 mM (data not shown). The values of the kinetic parameters determined for HIUHase are similar to the kinetic constants that characterize the preceding ureide pathway enzymes. Xanthine dehydrogenase has a K m for xanthine of 5 M and a turnover number of 5/s (11), and urate oxidase has a K m for urate of 10 M and a turnover number of 2/s (4).
The identification of HIUHase raises several important issues. First, the description of allantoin biogenesis remains incomplete. OHCU decomposes to allantoin, but for the same reasons discussed for HIU, a nonenzymatic pathway for allantoin formation does not seem likely. OHCU must undergo decarboxyation and tautomerization to form allantoin. In the nonenzymatic pathway a highly unusual 1,2-carboxylate shift leading to the formation of allantoin 5-carboxylate occurs (9). There is no reason to believe that this transformation occurs in a presumptive enzymatic pathway to allantoin formation, nor is there any evidence to rule out its occurrence. The results reported here suggest that a search for an enzyme that acts on OHCU is required, and we are undertaking that investigation.
Finally, the in vitro characterization we describe here, although suggestive, does not prove a role for HIUHase in the ureide pathway. Evidence for its in vivo role will come only from studies in which its activity is manipulated in planta using the techniques of molecular biology or the application of inhibitors. We have undertaken the cloning of the gene that encodes HIUHase, which we hope will facilitate investigations of its metabolic role.