**1. Introduction**

Ureases are metalloproteins responsible for the one step hydrolysis of urea into ammonia and carbamate [1], the later then rapidly and spontaneously decomposes to form carbon di‐ oxide and a second molecule of ammonia [2]. Plant ureases hold a special place in science history, participating on some important landmarks of biochemistry. For instance, it was by the analysis of *Canavalia ensiformis* urease crystals that the proteinaceous nature of enzymes could be demonstrated [3], rendering a Chemistry Nobel Prize to James Sumner in 1946. Al‐ so by the studies carried out with the same protein it was obtained the first proof that Ni2+ actually exerts a biological role in living organisms [4]. Ureases usually present two Ni2+ in their active site, with a few exceptions reported [5, 6]. However, all this was only possible after Takeuchi's observation that the crude extracts of soybean (*Glycine max*) seeds present high amounts of urease [7]. In those days, urease had been observed only in microorganisms and in algae, being Takeuchi's finding the first evidence of the presence of ureases in higher plants. The importance of this discovery relays on the fact that it made urease largely availa‐ ble for any researcher on the globe, and many more works on ureases followed, utilizing the soybean seed urease with the objective of understanding enzyme functioning. Soybean ure‐ ase was also one of the key players on the development of enzymology, with studies on this enzyme leading to hypothesis that were essential to confirm the observations of Michaelis and Menten on the rate of reaction of enzymes and substrates [8].

From those first studies more than one century ago until today, soybean ureases continued to be the focus of researchers around the world, in the fields of genetic, biochemistry and physiology. This review will deal with the many faces of these proteins, trying to summa‐ rize the great amount of information gathered over time, and to point the many doors that continue to be opened by the studies with this enzyme.

© 2013 Real-Guerra et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Real-Guerra et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. Soybean urease – more than one enzyme**

During the course of urease history, there were several reports on the presence of nongenet‐ ic isoenzymes for *C. ensiformis*, which represented different multimeric interconversible forms of the same enzyme. As many as 12 different forms of the protein could be observed, depending on environmental factors such as temperature, pH, salt concentration, reagents, etc [9-13]. Also, multimeric forms of the soybean urease were observed [14, 15]. By the com‐ parison of the purified urease from soybean seeds and a partially purified urease from a soybean shoot cell culture, it was not possible to differentiate them on basis of electrophoret‐ ic species, size exclusion chromatography and immunoinhibition, but they seemed to be‐ have differently on a immunoaffinity chromatography, where 100% of the seed enzyme would be retained in contrast with only 65 % of the culture urease [15]. Although the au‐ thors postulated that this behaviour may indicate the existence of two different enzymes, at the time they could not reject the hypothesis of differentially processed forms of the seed en‐ zyme (glycosylated protein, etc).

The next indication of the existence of more than one genetic isoform of soybean urease emerged a few years later, in 1982. After screening a collection of over 6,000 lines of soybean seeds from the United States Department of Agriculture (USDA) Polacco and co-workers were able to identify one urease negative soybean variety (Itachi) [16]. Interestingly, even though no measurable amounts of urease could be detected in the seeds of the Itachi variety, when cells from many parts of the plant were cultured, urease was produced in equivalent amounts to the wild type soybean. Making use of immunoaffinity chromatography with monospecific antibodies that retained 100% of the soybean seed urease, it was observed that none of the cell culture ureases could be retained completely by the antibodies (70 and 45 % for the wild type and Itachi, respectively). Some differences in the inhibition profile by hy‐ droxyurea were also observed, the ureases from both cultures (wild type and Itachi) being less susceptible than the urease purified from the seeds. All these facts pointed to a similar but still different urease synthesized in cell cultures as compared to the one present in the seeds. However, the authors seemed to battle a little with the idea of a second genetic iso‐ form of urease, considering that the observed "heterogeneity" could be due to a glycosylat‐ ed seed urease.

More definitive proofs of the existence of a second and distinct isoenzyme appeared when Kerr and co-workers [17] carried out biochemical characterizations of the seed and the leaf ureases, showing that the enzymes differed on several characteristics, such as optimum pH, apparent *K*m, inhibition profile by hydroxyurea and cross-reactivity against soybean seed urease antibodies. These observations were further extended by Polacco and Winkler [18] who found that the previously observed urease production by the cell cultures of a suppos‐ edly urease negative line actually corresponded to the production of a second isoform of urease present in the tested tissues (leaf, callus, seed and cotyledon). The hypothesis of the existence of more than one non-seed isoenzyme, very similar to each other but specific for each tissue, was considered at the time. Since then, this second isoform was denominated ubiquitous soybean urease (herein referred as ub-SBU) while the seed isoenzyme would lat‐ er be denominated embryo-specific urease (herein referred as es-SBU) [19].

The data gathered in these studies set ground for investigations in different fields and to the growing understanding of ureases, mainly in genetics, in the search for definitive answers regarding the diversity of this enzyme.

### **3. The discovery of soybean urease genes**

**2. Soybean urease – more than one enzyme**

zyme (glycosylated protein, etc).

Relationships

318

ed seed urease.

During the course of urease history, there were several reports on the presence of nongenet‐ ic isoenzymes for *C. ensiformis*, which represented different multimeric interconversible forms of the same enzyme. As many as 12 different forms of the protein could be observed, depending on environmental factors such as temperature, pH, salt concentration, reagents, etc [9-13]. Also, multimeric forms of the soybean urease were observed [14, 15]. By the com‐ parison of the purified urease from soybean seeds and a partially purified urease from a soybean shoot cell culture, it was not possible to differentiate them on basis of electrophoret‐ ic species, size exclusion chromatography and immunoinhibition, but they seemed to be‐ have differently on a immunoaffinity chromatography, where 100% of the seed enzyme would be retained in contrast with only 65 % of the culture urease [15]. Although the au‐ thors postulated that this behaviour may indicate the existence of two different enzymes, at the time they could not reject the hypothesis of differentially processed forms of the seed en‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The next indication of the existence of more than one genetic isoform of soybean urease emerged a few years later, in 1982. After screening a collection of over 6,000 lines of soybean seeds from the United States Department of Agriculture (USDA) Polacco and co-workers were able to identify one urease negative soybean variety (Itachi) [16]. Interestingly, even though no measurable amounts of urease could be detected in the seeds of the Itachi variety, when cells from many parts of the plant were cultured, urease was produced in equivalent amounts to the wild type soybean. Making use of immunoaffinity chromatography with monospecific antibodies that retained 100% of the soybean seed urease, it was observed that none of the cell culture ureases could be retained completely by the antibodies (70 and 45 % for the wild type and Itachi, respectively). Some differences in the inhibition profile by hy‐ droxyurea were also observed, the ureases from both cultures (wild type and Itachi) being less susceptible than the urease purified from the seeds. All these facts pointed to a similar but still different urease synthesized in cell cultures as compared to the one present in the seeds. However, the authors seemed to battle a little with the idea of a second genetic iso‐ form of urease, considering that the observed "heterogeneity" could be due to a glycosylat‐

More definitive proofs of the existence of a second and distinct isoenzyme appeared when Kerr and co-workers [17] carried out biochemical characterizations of the seed and the leaf ureases, showing that the enzymes differed on several characteristics, such as optimum pH, apparent *K*m, inhibition profile by hydroxyurea and cross-reactivity against soybean seed urease antibodies. These observations were further extended by Polacco and Winkler [18] who found that the previously observed urease production by the cell cultures of a suppos‐ edly urease negative line actually corresponded to the production of a second isoform of urease present in the tested tissues (leaf, callus, seed and cotyledon). The hypothesis of the existence of more than one non-seed isoenzyme, very similar to each other but specific for each tissue, was considered at the time. Since then, this second isoform was denominated Buttery and Buzzell [14] can be considered the pioneers on genetic studies involving soy‐ bean urease. In their work they identified in the soybean seed two variants of urease show‐ ing distinct electrophoretical mobility, which they assigned as fast- and slow-moving forms. These forms were later on characterized as the trimeric and hexameric forms of es-SBU, re‐ spectively [15, 19]. In their study, Buttery and Buzzell found the slow-moving form to be re‐ cessive, while the fast-moving form was considered dominant, and they concluded that a single locus (named *Eu*, after enzyme urease) controlled the expression of these electrophor‐ eticaly variant forms. Many years later, Kloth and Hymowitz [20] advanced the investiga‐ tion on the two multimeric forms and, by performing a series of crosses between urease null and wild type soybeans, proposed the existence of two codominant alleles, named *Eu1-a*, for the hexamer and *Eu1-b*, for the trimer. The *Eu1* gene, which encodes the es-SBU, was the first urease related gene described for a plant.

Probably the biggest milestone on soybean urease genetics research was the identification of null variants of es-SBU. First obtained by Polacco and co-workers [16], the variety Itachi was found to lack both the es-SBU protein [21] and mRNA [22]. Later on, four more mutants were identified [23], two lacking detectable amounts of es-SBU mRNA, and two producing very low protein levels. One of these later mutants produced a much altered protein. All those mutants were related to the locus *Sun* (for soybean urease null), and the *Sun* allele (normal es-SBU) was dominant over the *sun* allele (null es-SBU activity) [23-25]. The fact that allelic *sun* mutations affected urease transcript as well as es-SBU structure led to the conclu‐ sion that *Sun* and *Eu1* describe the same locus, and that the *Eu1-Sun* is the single functional es-SBU structural locus. Thus any mutant defective only on es-SBU is considered a *Eu1* or class I mutant. [25]. A genomic clone for urease was recovered from a soybean library and it was shown that this urease coding sequence was absent from seed urease-null mutants [22]. This would be confirmed as the first partial sequence obtained for the es-SBU years later.

Mutations obtained from ethyl methyl sulfonate treatment of soybean seeds [23] revealed a new class of mutants. Class II mutants produce normal levels of es-SBU and ub-SBU mRNA and protein, however their enzymatic activity is completely absent [23, 26]. Mutants of this class carry damage in one or both loci *Eu2* and *Eu3*, which are unlinked to each other or to *Eu1*. Studies on the urease profile during soybean development have shown that maximal synthesis of es-SBU in the developing cotyledon lags the maximal urease activity, suggest‐ ing a very slow maturation process of the enzyme *in vivo*. The facts that es-SBU is a Ni2+ containing enzyme [15] and the presence of Ni2+ is absolutely required for *in vivo* urease ac‐ tivity [27, 28] but not for urease synthesis [18, 21] are compatible with the assumption that Ni2+ emplacement into the active site could be a limiting process for urease activation. Hol‐ land and Polacco [29] have shown that class II mutants present normal Ni2+ uptake and translocation, thus eliminating the possibility that lack of access to Ni2+ was the reason for the absence of activity of both ureases. These observations pointed to *Eu2* and *Eu3* having a direct role on enzyme maturation probably by codifying proteins responsible for the Ni2+ emplacement, and that they are common for both es-SBU and ub-SBU [30]. As discussed lat‐ er, Eu2 and Eu3 were found to encode two different urease accessory proteins, involved in the enzyme maturation process.

After those findings, the remaining question was: is ub-SBU codified by a single gene or are there many tissue specific ureases? This question was clarified when mutants were obtained that presented normal levels of es-SBU activity, but no ub-SBU activity in any tissue. Those mutants were classified as class III mutants. Crosses between Class I and Class III mutants are devoid of ub-SBU even in embryonic tissues. The presence of the protein was detected in all tested tissues of class III mutants despite the lack of urease activity, showing that the le‐ sions affected directly the structural gene of ub-SBU resulting in the production of an inac‐ tive protein [26]. The lesions causing this effect were attributed to a new locus named *Eu4* [26, 31-33]. This finding demonstrated that the ub-SBU was actually a single protein synthe‐ sized in all tissues, and confirmed the existence of two genetic isoforms of soybean urease. A final blow on this question came when Goldraij and co-workers [34] finally obtained and se‐ quenced the cDNA of both isoforms showing that they are very similar, sharing 87% of identity and 92% of similarity.

The genomic era brought with it the last pieces of the puzzle. The sequencing of the soybean genome [35] confirmed the presence of the two previously described genes of urease, and also revealed a third one (Glyma08g10850), which is believed to be inactive due to a high number of deleterious mutations. Nevertheless, a residual activity was observed in double mutants, lacking both es-SBU and ub-SBU, and accounted for 2 to 10% of activity compared to the wild ub-SBU activity. This activity was designated as "background" and it was attrib‐ uted to microbial commensals in soybean tissues [26, 31, 36]. Alternatively, complementa‐ tion between the defective *Eu4* gene and this third urease-like gene may cause the residual activity [37]. The genome sequencing also brought light over the urease accessory proteins. A single gene for UreG, two for UreF and two for UreD were identified in the soybean ge‐ nome. Table 1 summarizes the data derived from the soybean genome available at the Phy‐ tozome databank [38].



**Table 1.** Urease-related genes in soybean

tivity [27, 28] but not for urease synthesis [18, 21] are compatible with the assumption that Ni2+ emplacement into the active site could be a limiting process for urease activation. Hol‐ land and Polacco [29] have shown that class II mutants present normal Ni2+ uptake and translocation, thus eliminating the possibility that lack of access to Ni2+ was the reason for the absence of activity of both ureases. These observations pointed to *Eu2* and *Eu3* having a direct role on enzyme maturation probably by codifying proteins responsible for the Ni2+ emplacement, and that they are common for both es-SBU and ub-SBU [30]. As discussed lat‐ er, Eu2 and Eu3 were found to encode two different urease accessory proteins, involved in

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

After those findings, the remaining question was: is ub-SBU codified by a single gene or are there many tissue specific ureases? This question was clarified when mutants were obtained that presented normal levels of es-SBU activity, but no ub-SBU activity in any tissue. Those mutants were classified as class III mutants. Crosses between Class I and Class III mutants are devoid of ub-SBU even in embryonic tissues. The presence of the protein was detected in all tested tissues of class III mutants despite the lack of urease activity, showing that the le‐ sions affected directly the structural gene of ub-SBU resulting in the production of an inac‐ tive protein [26]. The lesions causing this effect were attributed to a new locus named *Eu4* [26, 31-33]. This finding demonstrated that the ub-SBU was actually a single protein synthe‐ sized in all tissues, and confirmed the existence of two genetic isoforms of soybean urease. A final blow on this question came when Goldraij and co-workers [34] finally obtained and se‐ quenced the cDNA of both isoforms showing that they are very similar, sharing 87% of

The genomic era brought with it the last pieces of the puzzle. The sequencing of the soybean genome [35] confirmed the presence of the two previously described genes of urease, and also revealed a third one (Glyma08g10850), which is believed to be inactive due to a high number of deleterious mutations. Nevertheless, a residual activity was observed in double mutants, lacking both es-SBU and ub-SBU, and accounted for 2 to 10% of activity compared to the wild ub-SBU activity. This activity was designated as "background" and it was attrib‐ uted to microbial commensals in soybean tissues [26, 31, 36]. Alternatively, complementa‐ tion between the defective *Eu4* gene and this third urease-like gene may cause the residual activity [37]. The genome sequencing also brought light over the urease accessory proteins. A single gene for UreG, two for UreF and two for UreD were identified in the soybean ge‐ nome. Table 1 summarizes the data derived from the soybean genome available at the Phy‐

> **Protein codified**

**Protein size (aminoacids)**

urease <sup>839</sup> [23, 33, 34]

urease <sup>837</sup> [23, 33, 34]

**References**

the enzyme maturation process.

Relationships

320

identity and 92% of similarity.

tozome databank [38].

**Phytozome**

**accession id Gene (locus) Gene size (bp)**

Glyma05g27840 *Eu1* <sup>7736</sup> Embryo-specific

Glyma11g37250 *Eu4* <sup>7287</sup> Ubiquitous
