Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Abstract

Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the fivemember ring of allantoin (5-ureidohydantoin) to form allantoic acid. Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess ϳ1. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significant K i differences for the zinc and cobalt species (237 and 795 M, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.Allantoinase (EC 3.5.2.5), present in a wide variety of bacteria, fungi, and plants, as well as a few animals, such as fish and amphibians, catalyzes the conversion of allantoin (5-ureidohydantoin) to allantoic acid by hydrolytic cleavage of the five-member hydantoin ring (33, 52). Allantoin is a key intermediate in nitrogen metabolism of leguminous plants, such as soybean (Fig. 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. The product of allantoin hydrolysis, allantoate, is sequentially converted in soybean to ureidoglycine, ureidoglycolate, and glyoxylate with the ammonia released during these reactions utilized for plant cell growth. Variations of this pathway occur in other organisms, including Escherichia coli, which can grow on allantoin as the sole nitrogen source, but not as the sole carbon source. The E. coli allantoinase gene is part of a 12-gene regulon involved in allantoin degradation and nitrogen assimilation, and these genes are expressed only when the cells are grown under anaerobic conditions (10).On the basis of limited sequence similarities, allantoinase was suggested to belong to a broad family of metal-dependent amidohydrolases (17). Members of this family contain either mononuclear metal sites, such as the zinc site found in adenosine deaminase (56) and the iron site in cytosine deaminase (57), or dinuclear sites, such as the di-nickel site documented to occur in urease (20) and the di-zinc sites of dihydroorotase (47) and hydantoinase (1, 2, 8). Some, but not all, dinuclear hydrolase family members have their two metals bridged by a carbamylated lysine ligand (1,2,4,7,8,20,47). Significantly, we find that allantoi...

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

“…Note that, for functional analysis, the enzyme was expressed in the presence of zinc (Supplementary Data). 21 Mutation of Asn94 and Ser317, as well as the metal-ligating residue Asp315, completely abolished enzyme activity, and nonamended allantoinase, which was grown in the absence of supplemental zinc, remained inactive. In contrast, mutations at Arg215 and Ser288 did not exert significant effects on enzyme activity.…”

“…6 Biochemical studies using E. coli allantoinase have demonstrated that its activity depends on the presence and type of metal in the active center, analogous to other enzymes in this family. 20,21 Contrary to these observations, a metal-independent allantoinase (puuE) was recently characterized structurally and functionally from Pseudomonas fluorescens, in which allantoin was known to be used as a nitrogen source, even in the absence of gene for the metal-dependent allantoinase. 22 This new type of allantoinase was initially annotated as a polysaccharide deacetylase and has no sequence homology with the metal-dependent enzyme.…”

Functional Characterization of Allantoinase Genes from Arabidopsis and a Nonureide-Type Legume Black Locust

“…5, asterisks) responsible for metal biding were found in all reported ALNs. Recently, the recombinant E. coli ALN was shown to have the highest activity levels when observed in cell extracts from cultures supplemented with zinc and cobalt (Mulrooney and Hausinger, 2003).…”

Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase

“…The ion present in the enzyme is most likely to be manganese since it is the only cation that restored the activity in both enzymes, and cobalt has a predicted low bioavailability. The reconstitution of an enzyme in vitro by an ion different to that present in vivo is possible; cobalt is able to substitute other ions such as zinc (Mulrooney and Hausinger 2003) and manganese (Carvajal et al 1996). The effect of manganese is shared by other ureidoglycolate-degrading enzymes, such as ureidoglycolate urea-lyases from fish (Takada and Noguchi 1986) and microorganisms (Trijbels and Vogels 1967;Takada and Tsukiji 1987) and allantoicase from C. reinhardtii (Piedras and Pineda 2003).…”