**6. New features of an old protein – activities unrelated to the enzymatic one**

As stated above, no definite answer to the question of the physiological relevance of es-SBU has been given, since the demonstration that this enzyme plays no role in nitrogen assimila‐ tion from urea [32, 65, 73]. During the course of the last decade, a number of biological prop‐ erties unrelated to the enzymatic activity were described for plant ureases, launching a new look over these proteins and their physiological roles. Table 2 summarizes some of these overlooked biological activities of plant ureases. The main discoveries were made for the jackbean ureases (CNTX and JBU), revealing several interesting properties, such as entomo‐ toxicity [74-77], fungitoxicity [78, 79] and secretory activity [5, 80].

Some of the biological properties described for jackbean's ureases, such as the entomotoxic and fungitoxic activities, are shared by soybean urease. es-SBU displays toxicity toward in‐ sects, as demonstrated by [75]. es-SBU is toxic to *Dysdercus peruvianus*, a cotton culture pest, at doses as low as 0.05% (w/w), causing a decrease on insect body weight, delayed develop‐

ment and death. This toxicity was maintained after treating es-SBU with *p*-hydroxymercuri‐ benzoate (an irreversible urease inhibitor), confirming that the entomotoxic property was independent of the enzymatic activity. For the jackbean ureases, it has been demonstrated that this entomotoxic effect is a highly complex event, involving the intact protein as well as the release of a toxic peptide [82, 89, 90]. Jackbean ureases and their derived peptides affect several physiological processes in the insects, including the fluid transport across mem‐ branes [83, 91, 92]. So far, no studies regarding es-SBU mechanism of action in insects were performed, but it reasonable to assume that, due to their very similar sequences (92% of sim‐ ilarity), es-SBU and JBU would have the same targets in these organisms. The sequence of the entomotoxic peptide identified in CNTX and JBU, is present in es-SBU and ub-SBU [90]. These findings also support the hypothesis of a similar mode of action in insects.


**Table 2.** Properties of plant ureases unrelated to the enzymatic activity; N.D. = not determined; GHU = *Gossypium hirsutum* urease

Another very interesting property presented by es-SBU is its fungitoxic activity. es-SBU sup‐ pressed mycelial growth and/or inhibited spore germination of a series of fungi species and, as demonstrated for the entomotoxic property, this effect also does not require the protein's ureolytic activity [79]. The precise mechanism of action of ureases on fungi has not been elu‐ cidated so far, being proposed that ureases may interfere with the cellular osmotic balance. Recently, evidences of the participation of ub-SBU in soybean resistance to fungi were re‐ ported [93]. Soybeans mutants, lacking ub-SBU, were more susceptible to necrotrophic fun‐ gi, such as *Penicillium herguei*, *Phomopsis* sp and *Rhizoctonia solani*, and to the biotrophic pathogen *Phakopsora pachyrhizi*, responsible for the Asian soybean rust disease. In accord‐ ance to a defense role of ub-SBU against fungi infection, a rust-resistant soybean cultivar (PI561356) had a higher level of expression of ub-SBU after infection with *P. pachyrhizi*, in comparison with a susceptible cultivar (Embrapa-48) [93].

ment and death. This toxicity was maintained after treating es-SBU with *p*-hydroxymercuri‐ benzoate (an irreversible urease inhibitor), confirming that the entomotoxic property was independent of the enzymatic activity. For the jackbean ureases, it has been demonstrated that this entomotoxic effect is a highly complex event, involving the intact protein as well as the release of a toxic peptide [82, 89, 90]. Jackbean ureases and their derived peptides affect several physiological processes in the insects, including the fluid transport across mem‐ branes [83, 91, 92]. So far, no studies regarding es-SBU mechanism of action in insects were performed, but it reasonable to assume that, due to their very similar sequences (92% of sim‐ ilarity), es-SBU and JBU would have the same targets in these organisms. The sequence of the entomotoxic peptide identified in CNTX and JBU, is present in es-SBU and ub-SBU [90].

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

These findings also support the hypothesis of a similar mode of action in insects.

Entomotoxic

Relationships

326

Fungitoxic

Secretory

Toxicity to

*hirsutum* urease

*Rhodnius prolixus*, *Callosobruchus maculatus* [74], *Nezara viridula* [81], *Dysdercus peruvianus* [75, 76]

*Macrophomina phaseolina*, *Colletotrichum gloesporioides*, *Sclerotium rolfsii* [78]

Rabbit platelets [85, 86], rat brain synaptosomes [86], rat pancreatic cells [87], rat mast cells [88]

**Activity CNTX JBU es-SBU GHU**

*Dysdercus peruvianus* [75, 76, 82], *Rhodnius prolixus* [83] *Oncopeltus fasciatus* [77]

*Fusarium solani*, *Colletotrichum musae*, *Curvularia lunata*, *Penicillium herguei*, *Fusarium oxysporum* [79].

mammals 2 mg/kg (LD50) [5] Not toxic [75] Not toxic [75] N.D.

**Table 2.** Properties of plant ureases unrelated to the enzymatic activity; N.D. = not determined; GHU = *Gossypium*

Another very interesting property presented by es-SBU is its fungitoxic activity. es-SBU sup‐ pressed mycelial growth and/or inhibited spore germination of a series of fungi species and, as demonstrated for the entomotoxic property, this effect also does not require the protein's ureolytic activity [79]. The precise mechanism of action of ureases on fungi has not been elu‐ cidated so far, being proposed that ureases may interfere with the cellular osmotic balance. Recently, evidences of the participation of ub-SBU in soybean resistance to fungi were re‐ ported [93]. Soybeans mutants, lacking ub-SBU, were more susceptible to necrotrophic fun‐ gi, such as *Penicillium herguei*, *Phomopsis* sp and *Rhizoctonia solani*, and to the biotrophic

Rabbit platelets [5, 75] Rabbit platelets

*Dysdercus peruvianus* [75]

*Colletotrichum musae*, *Penicillium herguei*, *Curvularia lunata*, *Fusarium oxysporum* [79].

[75]

N.D.

*Colletotrichum musae*, *Penicillium herguei*, *Curvularia lunata* [84].

N.D.

These toxic activities, unrelated to the ureolytic one, are interesting findings that point to a possible role of ureases in plant defense against insects and fungi. However, plant ureases also have others bioactivities, that seem not related to plant physiology. JBU and es-SBU were shown to activate exocytosis in blood platelets causing them to aggregate, an effect shown to be independent of their enzymatic activity [75]. This exocytosis-inducing property may be relevant to some urease-producing microorganisms such as *Bacillus pasteurii* and *Helicobacter pylori* [94, 95] but it is unlikely to have a more specific significance for plants. Nevertheless, Carlini and Polacco [73] hypothesized that the secretion-properties of bacterial and plants ureases may play a role in rhizosphere relationships.
