Plant ureases : Roles and regulation

Both urea and urease were subjects of early scientific investigations. Urea was the first organic molecule to be synthesized and jack bean urease was the first enzyme ever to be crystallized. About 50 years later it was shown to be the first nickel metalloenzyme. Since then, nickel-dependent ureases have been isolated from many bacteria, fungi and higher plants. They have similar structures and mechanisms of catalysis. A urease apoenzyme needs to be activated. This process requires participation of several accessory proteins that incorporate nickel into the urease forming catalytic site. In this review, ureases from various organisms are briefly described and the similarities of their structures discussed. Moreover, the significance of urea recycling in plants is explained and recent literature data about the function and activation of plant ureases are presented.


ENZYMATIC ACTIVITY OF UREASE
Urease (EC 3.5.1.5,urea amidohydrolase), a nickel-dependent metalloenzyme, catalyzes the hydrolysis of urea to form ammonia and carbon dioxide.Hydrolysis of one molecule of urea results in the release of two molecules of ammonia and one molecule of carbon dioxide.
Several assays, mainly based on the measurement of the amounts of products released during the reaction, are available for quantify-ing urease activity.Ammonia can be detected by several methods including: ion-selective electrodes, reaction with phenol-hypochlorite or with Nesler's reagent.It is possible to include pH-sensitive dyes in the assays or simply observe changes with pH electrodes since release of ammonia results in an increase in pH.The amount of ammonia can also be monitored spectrophotometrically using a coupled system with NADH-dependent glutamate dehydrogenase (ammonia is a substrate for this enzyme).The other product of the reaction, carbon dioxide, can be trapped and monitored by radiological methods with 14 C-labeled urea as a substrate.For references and a more detailed review of the methods see (Mobley &Hausinger, 1989, andMobley et al., 1995).
Bacterial ureases play an important role in the pathogenesis of a number of bacterial species including Proteus mirabilis, Staphylococcus saprophiticus, Yersinia enterocolitica, Ureaplasma urealiticum and others (Mobley et al., 1995).Due to urease activity, bacteria (e.g.Klebsiella aerogenes) are able to use urea as a sole nitrogen source (Mulrooney et al., 1989).In the case of Vibrio parahaemolyticus, the ability to hydrolyze urea was proposed as a simple screening test to predict which strains are potentially pathogenic (Kaysner et al., 1994).One of the most frequently mentioned examples in the recent literature is the urease from Helicobacter pylori because of its essential role in the pathogenesis of this microorganism and the high prevalence of this human pathogen (Eaton et al., 1991).However, the best structural data are available for the urease from K. aerogenes (Jabri et al., 1995).
Urease activity was found in several species of fungi, however, the nucleotide sequences of the genes encoding urease were reported for only a few of them, including a fungal respiratory pathogen of human Coccidioides immitis (Yu et al., 1997) and Schizosaccharomyces pombe (Tange & Niwa, 1997).
In the invertebrate Aplysia californica, urease, together with carbonic anhydrase, is required for the formation and homeostasis of statoconia, calcium carbonate inclusions in the lumen of the gravity-sensing organ, the statocyst (Pedrozo et al., 1996a;1996b).
Urease is a cytosolic enzyme.In most of the studied cases (with exceptions that will not be discussed here) the majority of its activity is associated with the soluble fractions of the cells (Mobley et al., 1995).

PROTEIN STRUCTURE OF UREASES
The plant and fungal ureases are homo-oligomeric proteins (consist of identical subunits), while the bacterial ureases are multimers formed from a complex of two or three subunits (Mobley et al., 1995;Tange & Niwa, 1997).Significant amino-acid similarities were observed between all known ureases.Amino-terminal residues of the monomers of plant and fungal enzymes are similar to the small subunits of bacterial enzymes (e.g.UreA of H. pylori).The large subunits of bacterial ureases (e.g.UreB of H. pylori) resemble the carboxy-terminal portions of plant and fungal subunits (Fig. 1).The high sequence similarity indicates that all ureases 1190 A. Sirko and R. Brodzik are variants of the same enzyme and are likely to possess similar tertiary structures and catalytic mechanisms (Mobley et al., 1995).This conclusion is supported by the available biochemical and structural data obtained for the best-characterized ureases, e.g. from jack bean (Hirai et al., 1993) and K. aerogenes (Jabri et al., 1995).Jack bean urease exists as a homotrimer able to aggregate to a homohexamer (Hirai et al., 1993).Bacterial ureases possess structures similar to the jack bean urease.They are either trimers or hexamers of subunit complexes (Fig. 1).They can also exist in aggregated forms.The stoichiometry of subunits (1 : 1 for ureases from Helicobacter sp. or 1 : 1 : 1 for most bacterial ureases, including the urease from K. aerogenes) is always maintained.The catalytic site is located in subunit UreC of the K. aerogenes enzyme and in the respective regions (Fig. 1) of ureases from other organisms.
Each catalytic subunit contains the active site with two nickel ions that, in the case of crystallized K. aerogenes urease, were shown to be 3.5 Å apart (Jabri et al., 1995).In this enzyme, one nickel atom is bound to two histidine residues (His-246 and His-272), while the second nickel atom is bound to three residues: two histidines (His-134 and His-136) and aspartic acid (Asp-360).Additionally, a carbamate ligand derived from Lys-217 bridges the two nickel ions (Mobley et al., 1995).The process of nickel incorporation requires participation of many accessory (or activatory) proteins, which appear to act as urease-specific chaperones (Mobley et al., 1995).These proteins are required for assembling an active urease in both bacteria and plants (Polacco & Holland, 1994;Mobley et al., 1995).

ROLE OF UREASES IN PLANT METABOLISM
There are at least three key enzymes involved in urea metabolism in plants: arginase, urease and glutamine synthetase (Fig. 2).
The primary role of ureases is to allow the organism to use external or internally generated urea as a nitrogen source (Mobley & Hausinger, 1989;Mobley et al., 1995).Significant amounts of plant nitrogen flow through urea.This compound derives from arginine (Fig. 2) and possibly from degradation of purines and ureides (Polacco & Holland, 1994).The nitrogen present in urea is unavailable to the plant unless hydrolyzed by urease.The product of urease activity -ammonia -is incorporated into organic compounds mainly by glutamine synthetase (Fig. 2).It has been reported that overexpression of the pine glutamine synthetase in transgenic poplar improves the growth of the plants, probably by increasing the efficiency of nitrogen utilization (Gallardo et al., 1999).On the other hand, it has been shown that increased activity of this enzyme in the roots of a legume plant, Lotus japonicus, leads to decreased plant biomass production, possibly due to limited nitrate uptake (Limami et al., 1999).These results, which seem contradictory at first glance, might reflect the different roles of glutamine synthetase isoenzymes in different plants and/or different plant organs.They indicate the significance of this enzyme for plant nitrogen metabolism.The activity of glutamine synthetase, the amount of its product, glutamine, and possibly the availability of its substrate, ammonia, seem to be important factors controlling nitrogen metabolism and affecting plant growth (Stitt, 1999;Wiren et al., 2000).The ubiquitous urease (found in all plant tissues) is responsible for recycling metabolically derived urea (Polacco & Holland, 1994).In soybean, the ubiquitous urease is found at the level of 1/1 000 or 1/100 that of the embryo-specific urease.The latter does not seem to have any assimilatory function.Its physiological role is unknown, however, the involvement in protection against plant pathogens (due to production of toxic ammonia) has been proposed (Polacco & Holland, 1994).
As mentioned above, urea can be assimilated exclusively by urease in higher plants.Moreover, urease is the only nickel-containing metalloenzyme yet identified in plants (Polacco & Holland, 1993;1994).The importance of nickel for urease activity was demonstrated by the observation that urea-grown nickel-deprived rice (Oriza sativa) plants showed reduced growth and accumulated large amounts of urea due to reduced urease activity (Gerendas et al., 1998).Urease-negative mutant plants and nickel-deprived wild type plants have the same phenotype.They accumulate urea and exhibit necrotic leaf tips, apparently due to urea "burn" (Polacco & Holland, 1993).It has been demonstrated that similar leaf-tip necrosis observed after fertilization with urea result from the accumulation of toxic amounts of urea rather than from the formation of a toxic amount of ammonia (a product of urease action) since addition of a urease inhibitor increased the leaf-tip necrosis (Krogmeier et al., 1989).
Urease plays an important role in germination and in seedlings' nitrogen metabolism.It may function coordinately with arginase in the utilization of seed protein reserves during germination (Polacco & Holland, 1993).Imbibition of Arabidopsis seeds in water containing urease inhibitor delayed germination by 36 h and completely blocked germination of aged seeds.This inhibition could be abolished by supplying nitrogenous compounds into the imbibition medium (Zonia et al., 1995).

PLANT ACCESSORY PROTEINS INVOLVED IN UREASE MATURATION
Genetic analysis of urease expression in soybean revealed several genes whose inactiva- tion leads to the lack of urease activity (Polacco & Holland, 1993;1994).Two of these genes, Eu1 and Eu4, encode embryo-specific and tissue-ubiquitous ureases, respectively.Two other genes, Eu2 and Eu3, are analogous to bacterial urease accessory genes that are involved in urease maturation i.e. the placing of essential nickel in the urease active site.Mutation in either of the two latter genes eliminates the activity of the ubiquitous and embryo ureases but has little effect on the amounts of these proteins (Meyer-Bothling & Polacco, 1987; Meyer-Bothling et al., 1987).It was recently shown that Eu3 gene encodes the 32-kDa protein (Freyermuth et al., 2000).Its homologues have also been found in Arabidopsis (Freyermuth et al., 1999) and in Medicago truncatula (accession AA660998).Eu3 protein interaction with the product of Eu2 in the activation of embryo-specific urease was demonstrated (Freyermuth et al., 2000) but sequence data are not yet available for Eu2.
Freyermuth and coworkers have shown that the Eu3 protein is developmentally controlled and accumulates in developing embryos.They have proved the direct involvement of this protein in urease activation since anti-Eu3 antibodies blocked this activation in vitro (Freyermuth et al., 2000).Lack of Eu3 protein prevented accumulation of embryo-specific urease since the enzyme is most probably unstable without nickel insertion.Although the authors could not prove the direct involvement of Eu3 in the activation of tissue-specific urease (encoded by Eu4) because of the low sensitivity of the method (Freyermuth et al., 2000), such an involvement is rather expected, since both functional Eu3 and functional Eu2 are necessary for the activation of both types of ureases (Polacco & Holland, 1994).Urease activation in K. aerogenes involves the action of four accessory proteins (UreD, UreE, UreF and UreG) (Moncrief & Hausinger, 1997).These proteins are well conserved in bacteria that possess urease activity (Mobley et al., 1995).The Eu3 protein shows 55-60% identity in its C-terminal part to bacterial UreG proteins, including a nucleotide-binding P-loop that is essential for in vivo activation (Moncrief & Hausinger, 1997).The N-terminal part of Eu3 is rich in histidines and in this regard is similar to the C-terminus of K. aerogenes UreE (Freyermuth et al., 2000).No plant homologues of bacterial UreD, UreE or UreF have been yet identified.
In summary, several pieces of evidence suggest that both urease enzymes and the machinery required for their activation are evolutionary conserved.First, there is a significant sequence homology not only between urease enzymes (Fig. 1) but also between activatory factors (Freyermuth et al., 2000), although the latter is much less striking.Second, it was postulated that urease of soybean-commensal bacteria (Methylobacter) required some plant factors for activation (Polacco & Holland, 1994).Third, an increase of urease activity was observed in plants transformed with the ureA and ureB genes of H. pylori encoding subunits of urease (Brodzik et al., 2000).This result suggests that the plant accessory factors were able to incorporate nickel into a bacterial apoenzyme.The same enzyme produced in E. coli was inactive unless co-expressed with the H. pylori genes encoding accessory proteins (Hu & Mobley, 1993).

Significance of urea metabolism and advantages of increasing urease activity in plants
Nitrogen, after carbon, is the most limiting element in plant nutrition.Efficient recycling of reduced nitrogen present in the form of urea is important for plant growth since urea contains a significant amount of this element (Polacco & Holland, 1993).In addition to internally generated urea, externally applied urea can also be utilized by plants.Urea is a widely used fertilizer because of its low costs, ease in handling and high nitrogen content (Mobley & Hausinger, 1989).In plants, urease is the only enzyme that is able to recapture nitrogen from urea (Polacco & Holland, 1993).Fertilization with urea through leaves could be an efficient method of plant feeding and any modifications leading to increased urease activity in leaves could result in more effective assimilation of this fertilizer.Such an increase might have a positive impact on the nitrogen metabolism in plants since more ammonia would be available for assimilation via glutamine into a variety of nitrogenous compounds.

Figure 1 .
Figure 1.Schematic comparison of the structural subunits of ureases from selected organisms.The GenBank accession numbers (in brackets) and the numbers of amino acids (on the right) are shown for each protein.The percent values below boxes indicate the degree of identity to urease from jack bean (C.ensiformis).

Figure 2 .
Figure 2. Urea metabolism in plants.Modified from Gerendas et al. (1998).The names of the key enzymes involved in the production and conversion of urea are indicated in italics.