Importance of Urea Urease and Ni to Plants

Most or all plants have ureolytic activity (Hogan et al. 1982). To produce it they must deal with potentially toxic nickel (Ni), essential for an active urease. There are no other known plant Ni metalloenzymes (Gerendás et al. 1999) as opposed to six (urease, hydrogenase, CO dehydrogenase, methyl-CoM reductase, acetyl-CoA synthase and a superoxide dismutase) in bacteria (Ragsdale 1998). EST databases and mutational analysis of urease expression in soybean indicate that plants have specific permeases for Ni uptake and at least as many urease accessory genes as bacteria. Accessory genes encode proteins essential for insertion of Ni into apourease in vivo. It is worthwhile to ask why plants maintain this armamentarium for synthesizing urease and provisioning it with active site Ni. Just how important is the hydrolysis of urea to ammonia and CO2, and what is the source of urea?

Comparison of deduced and mature N-terminal AA sequences (Torisky et al. 1994) and localization analyses (Faye et al. 1985) indicate that ureases are cytoplasmic. Urease-negative soybean mutants grown with NH4NO3 or N2 as nitrogen (N) sources accumulate urea and exhibit necrotic leaf tip "urea burn" (Stebbins et al. 1991). Our evidence indicates that the metabolic source of all or most of this urea is arginine (Arg) (Stebbins, Polacco 1995). Arg is a pervasive N storage/transport compound in seeds, roots, tubers, bulbs, etc. In soybean seeds, Arg constitutes 18% of storage protein N (Micallef, Shelp 1989), and germination signals a massive conversion of liberated Arg to ornithine and urea via an inducible mitochondrial arginase (Goldraij, Polacco 1999). More germane to N fixation is the fate of ureides (allantoin and allantoate) exported from fixing soybean nodules. No more than a minor portion of the ureides was reported to be converted to urea in soybean plants (Stebbins, Polacco 1995; Winkler et al. 1987) and suspension culture (Stahlhut, Widholm 1989). It is worth considering, however, that some varieties may generate urea from ureides, and those that do so may have a greater ability to fix N under water stress. Soybean N-fixation is sensitive to water-deficit (Sinclair, Serraj 1995) under which leaf ureides accumulate (Purcell et al. 1998). Ureides have been shown to inhibit nodule nitrogenase (Serraj et al. 1999). Purcell et al. (2000) have proposed that sensitive varieties build up leaf ureides because leaf allantoate amidohydrolase (which generates ammonia and CO2 directly from allantoate) is less active due to reduced xylem delivery of manganese (Mn). Drought-tolerant varieties accumulate less ureide because they have an allantoate amidinohydrolase, which generates urea from allantoate and which does not require Mn. Mn stimulation of N fixation under water-deficit is consistent with this model as well as an earlier report (Shelp, Ireland 1985) that the drought-tolerant cultivar, Maple Arrow, produced urea from allantoate (Vadez, Sinclair 2000). While it is surprising that the presumed narrow germplasm base of North American soybean varieties could encompass two routes of ureide breakdown, urea producers (amidinohydrolases) are more common in nature. Recently, amidinohydrolases of ureidoglycolate (Piedras et al. 2000) and allantoate (Muñoz et al. 2001) were purified from Chlamydomonas and chickpea, respectively.

An active bacteroid hydrogenase, a Ni metalloenzyme, has been reported to improve the energetics of N fixation (Ruiz-Argiieso et al. 2000). Are Ni activation of hydrogenase and urease related? Olson et al. (2001) reported that the hydrogenase accessory proteins, HypA and HypB, were required for full activity of urease in Helicobacter pylori. We observed reduced activities of urease and hydrogenase of the phylloplane bacterium, Methylobacterium spp., when it colonized urease accessory gene mutants, eu2/eu2 and eu3/eu3, of soybean (Holland, Polacco 1992). Examination of urease may help define the defective step in the induction of hydrogenase activity in some Rhizobium leguminosarum-legume associations (Lopez et al. 1983).

Table 1. Comparison of soybean urease isozymes

Ubiquitous

Embryo

Tissue source

Embryo, seed coat, leaf,

Embryo

callus, roots

Crude seed extract Sp. Act.1

10"3 |imole/min x mg protein

1 |imole/min x mg protein

Subunit size2

95 kd

93.5 kd

Holomeric structure2

a3

CC3 (Eul-a) or 0C6 (Eul-b)

pH optima3'4

5.5, 8.8

7.5

Km

0.8 mM3

19-476 mM1

Hydroxyurea sensitivity5

Lesser

Greater

'Holland et al. 1987;2 Polacco et al. 1985;3 Kerr et al. 1983; 4 Torisky et al. 1984;5 Polacco, Winkler 1984; Polacco et al. 1982.

2. Urease: The Substrate of Urease Activation - in Plants, Bacteria, and Fungi

In soybean there are two distinct, non-allelic urease isozymes - the embryo (seed, embryo-specific) and the ubiquitous (metabolic, tissue-ubiquitous) - encoded by the Eul and Eu4 genes, respectively (summarized in Polacco, Holland 1994). Some distinguishing properties are presented in Table 1. The embryo has either an a3 or a6 structure, while the ubiquitous has been observed to be a3 (Polacco et al. 1985). Seemingly quite different is the (a,(3,y)3 structure of most bacterial ureases. However, these three distinct subunits are colinear with the single plant (and fungal [Figure 1]) subunit. Klebsiella and Jack bean ureases have >50% AA identity along their aligned regions (Mulrooney, Hausinger 1990).

Ardbidopsis, 840 AA Jack bean 838 AA

5 pombe, 835 M

K/ebsfef/a, 1DD+1Q7+567 AA

5 pombe, 835 M

K/ebsfef/a, 1DD+1Q7+567 AA

Figure 1. Subunit structure of bacterial and eukaryotic ureases and the AA identity over their co-aligned regions. S. pombe urease has a single 95 kDa subunit, but with sequence identity between the bacterial and plant ureases. Identities were determined with DNASIS 2.5 software (Hitachi Software Engineering Corp., Yokohama).

The relatedness of a fungal (Schizosaccharomyces pombe) urease (Tange, Niwa 1997) to two plant ureases is illustrated in Figure 1. Jack bean urease is the embryo isozyme. The tissue distribution and metabolic function Arabidopsis urease (Zonia et al. 1995) indicate that it is the ubiquitous type. Limited sequence data for the soybean ureases indicate a high degree of relatedness of both isozymes to the Jack bean embryo urease (Torisky et al. 1994; Krueger et al. 1987). Our working hypothesis is that correctly identified accessory proteins from soybean and Arabidopsis can activate the related, plant-like urease of S. pombe. Table 2 indicates that soybean contains two accessory genes each of which activates both urease isozymes.

3. Urease Accessory Gene Candidates in Soybean

Table 2 summarizes our knowledge of the genes controlling the urease activities of soybean. Since the structural genes are fusions of two or three bacterial genes, we reasoned that the number of accessory genes could be reduced as well if the bacterial counterparts were represented as domains on fused plant proteins. This hypothesis is appealing not only because we would have fewer genes to characterize, but also because complexes among bacterial accessory proteins have been demonstrated. Bacterial urease clusters contain, other than 2 or 3 structural genes plus Ni transporters, a maximum of 4 accessory proteins: UreD- a chaperone-like protein, UreE- a Ni-binding protein with a His-rich C-terminus, UreF- essential for proper insertion of Ni in the active site, and UreG- probably a GTPase. UreD, UreF and UreG form a complex that interacts with apo-urease. Each is essential for an active urease, in vivo. UreE deletions have about 50% normal urease activity, but Ni supplementation can increase this basal level. Hausinger's lab has been in the forefront of advancing our knowledge of bacterial urease activation (see Hausinger, Karplus 2001 for a review).

Table 2. Genes controlling urease production in soybean

UREASE ACTIVITY IN MUTANT

LOCUS

+

FUNCTION Structural (embryo)1'2 Structural (ubiquitous)1

Eu2 Eu3 Eu5

Accessory (UreG) 3'4

Accessory

'Meyer-Bothling, Polacco 1987; 2Torisky et al. 1994; 3Polacco et al. 1999; 4Freyermuth et al. 2000.

'Meyer-Bothling, Polacco 1987; 2Torisky et al. 1994; 3Polacco et al. 1999; 4Freyermuth et al. 2000.

4. Soybean Eu3 Encodes a UreG-like Protein with Ni-binding Capacity

EST databases of several plants identified UreG, though we first cloned it serendipitously. This is the best conserved accessory gene in bacteria and appears to be well conserved in plants, that of Arabidopsis and soybean sharing 80% identity and 88% similarity. By exploiting plant UreG cDNAs, we conclude that soybean Eu3 encodes the UreG ortholog: (1) The eu3-el/eu3-el null mutant lacks UreG antigen, mRNA and a genomic EcoRV fragment with UreG homology. (2) A dominant, leaky allele, Eu3-e3, contains an alanine to valine substitution. (3) Anti-UreG inactivates the Eu3 gene product in in vitro activation of urease in mixed eu2/eu2 + eu3-el/eu3-el developing embryo extracts (Freyermuth et al. 2000). Soybean UreG binds to a Ni column, as expected from a His-rich N-terminus, exclusively found in plant UreGs (Witte et al. 2001). This His-run led us to consider a UreG-UreE fusion at first, especially since UreE has yet to be reported in any plant database.

5. UreG Complementation Across Kingdoms

The UreG sequence is well conserved between bacteria and plants. Hence it is not too surprising that potato UreG was able to complement, albeit at low efficiency, a UreG deletion in the Klebsiella operon (Witte et al. 2001). This occurred in spite of the His-rich N-extension in the plant UreG. We observed no Arabidopsis UreG complementation of a UreG disruption of R. leguminosarum.

The greater similarities in both UreG and urease (Figure 1) between S. pombe and plants lead to a greater expectation for plant UreG to function in S. pombe.

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