Cloning and Expression of the Gene for Soybean Hydroxyisourate Hydrolase. Localization and Implications for Function and Mechanism

Abstract

The gene encoding hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme, has been cloned from soybean (Glycine max). The gene encodes a protein that is 560 amino acids in length and contains a 31-amino acid signal sequence at the N terminus that is not present in the mature protein. The presence of two SKL motifs near the C terminus suggests that the protein resides in the peroxisome. This expectation is borne out by results from immunogold electron microscopy, which revealed that hydroxyisourate hydrolase was localized in the peroxisomes of uninfected root nodules. The gene encoding hydroxyisourate hydrolase was expressed in Escherichia coli, and soluble, catalytically active enzyme was purified to homogeneity. Sequence analysis revealed considerable homology with members of the ␤-glucosidase family of enzymes. Two glutamate residues, E199 and E408, align with the conserved glutamates that play catalytic roles in the ␤-glucosidases. However, the other residues that have been identified by crystallography to interact directly with the substrates in ␤-glucosidases are not conserved in hydroxyisourate hydrolase. The E199A and E408A hydroxyisourate hydrolase mutants were devoid of detectable catalytic activity. Analysis of transcripts for hydroxyisourate hydrolase demonstrated that its level of expression was highest in the nodule; mRNA was detectable 12 d after infection and increased until 21 d postinfection, then declined. In a similar manner, immunodetection of hydroxyisourate hydrolase indicated preferential localization in the nodule; the amount of protein detected was maximal at 21 d postinfection. The pattern of expression of hydroxyisourate hydrolase matched that of urate oxidase, and supports the hypothesis that hydroxyisourate hydrolase plays a role in ureide metabolism.Nitrogen is frequently the limiting nutrient for growth of many crop plants (Schubert, 1986), and tropical legumes such as soybean (Glycine max) obtain the nitrogen that ultimately supports protein synthesis via a particularly sophisticated system. The plants establish a symbiotic relationship with bacteria from the genus Bradyrhizobium, which contain the enzyme nitrogenase. Nitrogenase catalyzes the conversion of atmospheric dinitrogen to ammonia, which can be used to meet the plant's metabolic nitrogen requirements, but first it must be converted into organic forms that can be transported throughout the plant and further metabolized. Isotope-labeling studies demonstrate that ammonia produced from dinitrogen reduction is rapidly converted into allantoin and allantoate, the so-called ureides (Ohyama and Kumazawa, 1978); up to 95% of the nitrogen in the xylem sap in nodulated soybeans is in the form of ureides (Schubert, 1981).The ureides are derived from the oxidation of purines, and allantoate arises from the hydrolysis of S-allantoin, but details of the origin of allantoin remain unclear. It has been widely reported that allantoin is the product of the urate oxidase (UO) reaction, but recent work has established that UO catalyze...

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).…”

Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

“…Of these, GH1 has been most thoroughly documented and shown to comprise a gene family encoding 40 putative functional GHs in Arabidopsis (Arabidopsis thaliana) and 34 in rice (Oryza sativa) in addition to a few pseudogenes (Xu et al, 2004;Opassiri et al, 2006). In addition to b-glucosidases, plant GH1 members include b-mannosidases (Mo and Bewley, 2002), b-thioglucosidases (Burmeister et al, 1997), and disaccharidases such as primeverosidase (Mizutani et al, 2002) as well as hydroxyisourate hydrolase, which hydrolyzes the internal bond in a purine ring rather than a glycosidic bond (Raychaudhuri and Tipton, 2002). The specificity for the glycone in GH1 enzymes varies.…”

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

“…The first step in this pathway is the degradation of nucleic acid purine moieties (adenine and guanine) to uric acid. After two consecutive oxidation reactions by urate oxidase and hydroxyisourate hydrolase, the uric acid is converted to ALN (Raychaudhuri and Tipton, 2002). Allantoinase (ALN amidohydrolase, EC 3.5.2.5) catalyzes the hydrolysis of ALN to form allantoic acid, which is a key reaction for biogenesis and the degradation of ureides (Vogels et al, 1966;Noguchi et al, 1986).…”