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

Abstract

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 ϳ100-fold to apparent homogeneity with a final specific activity of 10 mol/ min/mg. The enzyme exhibited a native molecular mass of ϳ68 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 the K m for 5-hydroxyisourate was determined to be 15 M. 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 fi...

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

“…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 oxidase (9) but differ in that racemic allantoin is produced in the nonenzymatic decomposition of HIU (10).…”

Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family

“…Tipton and colleagues have provided details of the urate oxidase-catalyzed reaction (5,6) and subsequently identified a gene for HIUHase from soybean (G. max) (7,8). The 560-aa soybean HIUHase does not share any sequence similarity with PucM, but belongs to family 1 of glycosidase (9).…”

“…1.7.3.3; uricase), but recent investigations have revealed that this pathway includes two additional, distinct, chemically labile intermediates: 5-hydroxyisourate (HIU) and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) (5,6). It is now generally believed that urate oxidase is responsible only for the conversion of uric acid into HIU, and a second enzyme, HIU hydrolase (HIUHase), catalyzes the hydrolysis of HIU into OHCU (7)(8)(9)(10)(11), which is then converted into allantoin via an enzyme-dependent decarboxylation reaction (Scheme 1) (11). Recently, three genes were characterized as HIUHases: a soybean (Glycine max) gene (7,8), PucM in Bacillus subtilis (10), and MuraH in mice (11).…”

“…It is now generally believed that urate oxidase is responsible only for the conversion of uric acid into HIU, and a second enzyme, HIU hydrolase (HIUHase), catalyzes the hydrolysis of HIU into OHCU (7-11), which is then converted into allantoin via an enzyme-dependent decarboxylation reaction (Scheme 1) (11). Recently, three genes were characterized as HIUHases: a soybean (Glycine max) gene (7,8), PucM in Bacillus subtilis (10), and MuraH in mice (11). Sequence alignment has shown that the PucM and MuraH genes belong to the same family of proteins, but the third gene differs markedly from the other two in size and sequence similarity (11).…”

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

“…1), where ureides provide the major transport form of symbiotically fixed dinitrogen (29,43,48,55). Until recently, it was thought that allantoin was formed directly from uric acid by the action of uric acid oxidase (39); however, it has now been established that the true product of soybean uric acid oxidase is 5-hydroxyisourate (22), which is then acted upon by the enzyme hydroxyisourate hydrolase to yield 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (42). This compound is then converted to allantoin, most likely by a yet unidentified enzyme.…”