Occurrence and function of ureases

Ureases are widespread in nature, are synthesized by numerous organisms and are also present in soils as a soil enzyme (see below). The substrate urea is readily available, arising mainly from urine excretion by animals, from the decomposition of N- compounds from dead organic matter (Wang et al., 2008), or from its application as a fertilizer. Thus ureases play a prominent role in the overall nitrogen metabolism in nature where their key function is to provide organisms nitrogen in the form of ammonia.

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1.4.1. Soil urease and ammonia volatilization

Of great importance in agriculture is the ureolytic activity of soils (Krogmeier et al., 1989; Mulvaney, 1981) which is derived maily from soil urease rather than from microorganisms (Mulvaney, 1981). The urease enzyme in residues of dead plant and microbial cells becomes extracellular and is highly stable thanks to the immobilization on clays and humic substances (Krajewska, 2009). The presence of this stable form of urease in soils allows urea to be used as an efficient nitrogen fertilizer. Due to its high nitrogen content, chemical stability and low cost in production, urea makes up over 50% of the total nitrogen fertilizer applied worldwide. The role of soil urease is to hydrolyse the urea to ammonia to make it available to plants. However, if the hydrolysis is too rapid it may result in unproductive loss of nitrogen by ammonia volatilization, while ammonia toxicity and alkalinity along with nitrite accumulation may induce damage to plants, thereby causing severe environmental and economic problems (Krogmeier et al., 1989; Mulvaney, 1981). Ammonia volatilization also causes problems in the management of livestock waste where the loss of nitrogen in the livestock slurry leads to a reduction in its value as fertilizer, and the source of pollution, ammonia, contributes to the adverse odour. Attempts have been made to recycle urine to use as flush water by suppressing urease activity to avoid ammonia emission (Ikematsu et al., 2007). In medical, agriculture and environmental settings where it is important to control the urease activity, the use of urease inhibitors has been proposed to avoid its negative effects (Krajewska, 2009).

1.4.2. Urease in plants

The first plant urease gene has been characterized from soybean (Glycine max). The soybean genome contains an embryo-specific urease encoded by the gene Eu1 (Meyer-Bothling et al., 1987) and a ubiquitous urease encoded by Eu4 (Torisky et al., 1994). The residual urease activity in Eu1/Eu4 double mutants was explained by urease-producing bacteria living on the plant (Holland and Polacco, 1992). In contrast, potato (Solanum tuberosum), tomato (Lycopersicon esculentum) and other solanaceous species, as well as Arabidopsis thaliana possess only a single urease gene (Witte et al., 2005).

125 The urease enzyme plays an essential role in catalyzing urea assimilation after uptake into the plant cell (Wang and Köhler, 2008; (Kojima et al., 2006). Higher plants posses various urea transport systems, both passive and active, which allow them to optimize nitrogen assimilation depending on the form available in the environment or internally. Although plants can assimilate nitrogen in form of urea though the roots, uptake occurs mostly in the form of ammonia which generated from urea hydrolysis though soil ureases.

Besides hydrolyzing the urea acquired from the environment, ureases allows plants to recycle nitrogen from urea originating from two metabolic processes: the arginase-catalyzed breakdown of arginine (Zonia et al., 1995) and the degradation of purines and ureides (Todd et al., 2006; Winkler et al., 1988). However plants have the capacity to degradation purine and ureides without generating urea intermediate, leaving arginine catabolism as the only confirmed source of urea (Witte, 2011).

Because nitrogen availability is generally growth-limiting for plants (Bray, 1983), efficient recycling is likely to provide plants an ecological advantage. Urea is metabolized rapidly and does therefore not accumulate, however when constantly generated it may serve as a nitrogen source. Combined genetic and biochemical analyses revealed that urease enzyme activity is regulated by the global nitrogen regulatory system (Magasanik, 1988) acting through the nac (nitrogen assimilatory control) gene product (Bender et al., 1983, Macaluso et al., 1990). Under conditions of low nitrogen availability, urease activity is induced. Interestingly, it was suggested that besides their ureolytic enzyme activity, plant ureases may play a role in the plant defence system because urease exhibits insecticidial (Follmer et al., 2004) and antifungal properties (Becker-Ritt et al., 2007; Menegassi et al., 2008).

1.4.3. Metabolic sources and transport of urea in plants

In plants, urea is especially important during germination and originates from the breakdown of arginine (Zonia et al., 1995) and from purines or ureides (Todd et al., 2006; Winkler et al., 1988). Arginine is the most important single metabolite for nitrogen storage in plant seeds (Vanetten C.H, 1967) and its catabolism is central to the mobilization of nitrogen from tissues. The importance of urease for recycling arginine nitrogen during germination is highlighted by the fact that aged Arabidopsis seeds failed to germinate when urease was chemically

126 inhibited but could be rescued by an external nitrogen source (Zonia et al., 1995). First, mitochondrial arginase hydrolyses arginine to access the stored nitrogen in the guanidinium group, thereby generating ornithine and urea which are then converted by the mitochondrial ornithine metabolism to glutamate (Funck et al., 2008). Urea is exported to the cytosol by a passive transport, possibly through aquaporins (Soto et al., 2010) also called MIPs (major intrinsic proteins), which conduct selected low molecular solutes along a concentration gradient through a channel. In the cytosol urea is hydrolysed by urease, and the urea-derived ammonium is re-assimilated by cytosolic glutamine synthetase, using glutamate from ornithine catabolism as the substrate.

Through these reactions, all the nitrogen from arginine is incorporated into glutamine, while urease is required to mobilize half of the nitrogen stored in arginine (Figure 2). This is the only firmly established role of urease in plant metabolism, apart from hydrolyzing root- imported urea, and arginase is the only plant enzyme known to generate urea in vivo. In the second pathway, urea is produced by the catabolism of purines or ureides like allantoin and allantoate, which are used for example by leguminous species for long-distance translocation of nitrogen (Stebbins and Polacco, 1995).

However, urea does not only originate from arginine or purine breakdown but can also be taken up from the environment via urea transporters (Kojima et al., 2006) Wang et al., 2008). Plants possess a high affinity urea transporter (DUR3) that is involved in uptake of environmental urea while also mediating internal urea transport. In A. thaliana DUR3 was identified by its similarity to the urea transporter of S. cerevisiae (ScDUR3) (Liu et al., 2003). The protein is localized at the plasma membrane of root epidermal cells especially in nitrogen starved plants, and the gene expression is induced by urea in the absence of other nitrogen sources (Kojima et al., 2007; Merigout et al., 2008). A role of Dur3 in internal urea transport is indicated by the expression of AtDUR3 near the root xylem and in the shoots (Kojima et al., 2007; Liu et al., 2003).

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