Ureases: Functions, Classes, and Applications

Ureases: Foundations, Classes, and Applications provides a thorough, practical analysis of ureases—enzymes of growing relevance across a range of biotechnological applications and drug discovery. Unique in many aspects, ureases are one of the few enzymes to have nickel in their active sites. Ureases covers all aspects of this enzymatic class. Starting with foundational overview, the book discusses historical urease research and the current state, from basic biochemistry to the use of ureases as hallmarks in enzymology, crystallography, and bioinorganic chemistry. The different classes of ureases, structurally diverse but chemically equivalent, are individually discussed. The multi-protein, multi-step activation of ureases (with chemical modification of residues, transport, and transfer of nickel ions) are examined in-depth, along with the catalytic mechanisms of ureolysis and its inhibitors. The final two sections of the book address multiple applications of ureases in health and biotechnology, respectively, going from gastric ulcer treatment to architectural uses in buildings and engineering. Future applications and next steps in research are also considered.

• Considers fundamental aspects of urease biochemistry, ureolysis and urease inhibitors
• Discusses urease research across a range of applications, from drug discovery to biosensors, water purification, architecture and the food industry
• Features chapter contributions from international leaders in the field

• Language: English
• Publisher: Academic Press
• Release date: Feb 16, 2024
• ISBN: 9780323972000

Book preview

Preface

Urease! What a picture the word

conjures up for all those who have had

even the most fleeting flirtation with

chemistry.

B. Zerner

While preparing this book, its two editors faced their own trajectories with urease. Oddly enough, despite being a poster child for enzymology, it was not the catalytic role that led the way in the editors’ long relationship with ureases (indeed, way more than a mere flirtation, in the words of Zerner).

Célia Carlini stumbled upon ureases while trying to identify a novel toxin isolated from legumes. Involuntarily, a whole new set of biological activities was ascribed to ureases, reinforcing its multiple roles as a virulence factor. Later on, under Célia’s guidance, Rodrigo Braun started exploring evolutionary explanations for various structural idiosyncrasies observed in ureases, from their unnecessarily large sizes to requiring an arsenal of accessory proteins for proper activity.

Not being urease experts from the get-go, they had to make up for the accumulated data on this enzyme. Their sinuous path connected multiple researchers worldwide, whose significant contributions we are thankful for collating in this volume. Apart from being a tentative reference tome on ureases, this book embodies decades of collaborations, discussions, innovations, and lives dedicated to a single biomolecule.

The need for completeness, although unrealistic, haunted the chapter organization. Here are introductions to urease, its history, and personal recollections of scientists directly involved in such research. Urease structure, activity, activation, and inhibition are all discussed, along with urease involvement in health and disease. Multiple applications of ureases and advantages in harnessing their numerous biological activities are presented. Surely, there is always room for improvement, and some chapters that, for one reason or other, had to be reshaped or scrapped along the way are sorely missed.

The editorial process started during the COVID-19 pandemic, in a moment of great uncertainty in science and everyday life (they may be the same, after all). Thus we are indebted to all authors who contributed their work to this book (and put on with our multiple deadline changes). Likewise, we are thankful to the entire Elsevier team, especially Michaela Realiza, for managing the project with the proper balance between kindness and keenness.

Lastly, we hope you, dear reader, find ureases as compelling as we all do. Theirs is a very long and convoluted story, but far from over. We do hope you are the one to show us all the next breakthrough in the chronicle of this old friend.

Rodrigo and Celia

Part I

Introduction

Outline

1 Ureases: an overview

1

Ureases: an overview

Conrado Pedebos¹ and Rodrigo Ligabue-Braun²,    ¹Graduate Program in Biosciences (PPGBio), Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil,    ²Department of Pharmacosciences, and Graduate Program in Biosciences (PPGBio), Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil

Abstract

Ureases perform the hydrolysis of urea to ammonia and carbonic acid. As they are highly prevalent and proficient, they have similarities in different levels of protein structural hierarchy. Their rare requirement for nickel in their active sites is associated with a series of proteins responsible for proper active site assembly and metal delivery. Ureases are also moonlighting proteins, having multiple biological activities that are unrelated to catalysis. Such activities are combined with urea production in pathogenic organisms such as Helicobacter pylori, Cryptococcus spp., and Proteus mirabilis. The enzyme also has industrial applications in biosensor manufacturing, beverage detoxification, and biocement production. This chapter is intended as a primer on ureases, summarizing the aspects that are explored in depth in the remainder of this volume.

Keywords

Urease; ureolysis; urea; infection; agriculture

1.1 Introduction

Ureases are enzymes of the urea amidohydrolases class (E.C. 3.5.1.5) that perform urea hydrolysis, leading to the formation of ammonia and carbonic acid¹ (Fig. 1.1). They are found among multiple taxa, including plants, fungi, eubacteria, and archaea, with the exception of animals.² The pervasive presence of environmental urea is taken as the driving force for the evolution of ureases. In comparison to the spontaneous urea decomposition by elimination, the urea hydrolysis performed by ureases is considered to be an acceleration in the reaction rate by a factor of 10¹⁴. For this reason, urease is considered the most proficient enzyme known to date.³

Figure 1.1 General representation of the urease-catalyzed reaction of urea hydrolysis.

1.2 Structure and activation

Structurally, all ureases share the same features.⁴ They all have a functional monomer architecture that can be a true monomer (in plants and fungi) or an oligomer formed by two (some prokaryotes) or three heteromonomers (majority of prokaryotes) homologous to the structural domains in the true monomer. These functional monomers are further organized into trimers, hexamers, or dodecamers³ (Fig. 1.2). The tridimensional organization of ureases is unique among enzymes.⁶ The high amino acid sequence similarity (55% identity) among ureases is taken as evidence for their divergence from a common ancestral protein, with the closest relative of ureases being dihydroorotase.⁷ A previous phylogenetic reconstruction suggested that tri-chained ureases became fused as monomeric ureases in a horizontal gene transfer followed by readthrough of stop codons.⁶ Di-chain ureases are special cases in this proposition, not being an intermediate between trimers and monomers (all of them corresponding to the urease functional monomer). Still, the reassessment of urease phylogenies suggests a more complex scenario that may require further inspection.⁸

Figure 1.2 Structural conservation and supramolecular arrangements in ureases (Sporosarcina pasteurii urease, PDB ID 2UBP; Helicobacter pylori urease, PDB ID 1E9Z; Canavalia ensiformis urease, PDB ID 3LA4; geometric representation based on Tsang and Wong⁵).

The first report of nickel in any catalytic center was in urease in 1975.⁹ To date, there are less than a dozen other occurrences of nickel as part of biomolecules. Interestingly, that is not the only reason to consider ureases as hallmarks in biochemistry. In 1926 it was shown for the first time that enzymes are proteins (a very controversial proposition at the time), and it was done by the crystallization of urease.¹⁰

As a dinuclear metallohydrolase, ureases are considered unique by being Ni(II)-dependent.¹¹ The metal centers in the active site activate urea and water allowing for hydrolysis. Besides the two nickel atoms (rare exceptions are iron-containing ureases in organisms associated with carnivory),¹² the urease active site is composed of four histidines, one aspartate residue, one carbamylated lysine, and a water cluster¹³ (Fig. 1.3). A mobile flap (a helix-turn-helix structural motif) that controls substrate entrance and product exit from the active site is also considered part of the active site.⁴

Figure 1.3 Structural depiction of urease active sites (based on Sporosarcina pasteurii urease, PDB ID 2UBP).

The structural specificity of urease active site requires a series of steps for assembly and proper functionality, a process known as urease activation or maturation.¹¹,¹⁴ These steps pertain mostly to nickel transport and delivery as well as lysine modification.

In bacteria, urease activation involves four accessory proteins (UreD, UreF, UreG, and UreE). The genes encoding these proteins are almost always organized along with urease genes (for subunits UreA, UreB, and UreC) in an operon, and, apart from UreE, all others are shown to be essential for proper ureolytic capacity. Urease activation can be briefly described as follows⁵,¹⁵ (Fig. 1.4): the apo-urease oligomer UreABC binds to UreD, considered to be a scaffold for the further activation complex organization. Then, UreF binds UreABC-D, acting as a GTPase-activating protein. UreG (an intrinsically disordered enzyme) acts as GTPase. With GTP hydrolysis, UreE delivers the metal ions to the UreABC-UreDFG oligomer. All urease accessory proteins are metallochaperones, since they are involved in the transport of nickel ions. In plants and fungi, UreG and UreE are found as a single protein, with UreG carrying a histidine-rich N-terminal that mimics UreE nickel-binding abilities.

Figure 1.4 Urease activation pathway, highlighting the multiple steps, multiprotein complexes required for proper enzymatic activity (based on Tsang and Wong,⁵ Nim et al.¹⁵).

The mechanism of urease-catalyzed urea hydrolysis has been a contentious topic. Nowadays, mostly due to inhibitor-based studies, there seems to be an agreement about its steps.¹⁶ Urea replaces three water molecules in the urease active site, binding to Ni(1) via the carbonyl oxygen, the urea carbon becomes more electrophilic (increased susceptibility to nucleophilic attack) and then urea binds to Ni(2) by one of its N atoms, forming a bidentate bond with the enzyme. This bond facilitates the nucleophilic attack of the carbonyl atom by water, forming a tetrahedral intermediate that releases NH3 and carbamate.

1.3 Relevance in health and technology

Ureases are relevant in health and (bio)technology. Health impacts of urease-dependent organisms include stomach, brain, and kidney infections¹⁷ (Fig. 1.5). Ureases are also considered moonlighting toxins, since they have many biological activities that are unrelated to their catalytic one,² something that impacts their relevance as virulence factors.

Figure 1.5 Health-related urease activities in human patients.

Arguably the most relevant (or, at least, the most famous) role of urease in disease is its association with gastritis, peptic ulcer, and gastric cancer.¹⁸ H. pylori, an acid-resistant Gram-negative bacterium, is able to colonize the harsh stomach environment by producing an enormous amount of urease. This urease, by producing ammonia, is able to raise the pH in the bacterial microenvironment, thus making the acidic environment not only tolerable but also thriving for H. pylori.¹⁹ Despite having very well-known virulence factors associated with the mucosal colonization and its modification toward cancerous tissue, there is growing evidence that urease plays a role beyond alkalization.² Also, in the infection context, urease is a virulence factor for Proteus mirabilis and Cryptococcus spp. The P. mirabilis is a Gram-negative bacterium that is commonly found in soil, water, and the digestive tract of humans and other animals. While it is generally considered harmless, it can cause urinary tract infections (UTIs) in individuals with weakened immune systems or structural abnormalities in the urinary tract.²⁰ P. mirabilis uses its urease activity to create its own niche within the urinary tract, allowing it to colonize the bladder and kidneys and cause infection. In the urinary tract, the alkalinization of urine can lead to the formation of kidney stones, by facilitating the precipitation of carbonates, creating an ideal environment for bacterial growth.²¹ In addition, the ammonia produced by the breakdown of urea can be toxic to the cells lining the urinary tract, leading to tissue damage and inflammation.

Cryptococcus is a genus of fungi that can cause life-threatening infections, particularly in individuals with weakened immune systems.²² One of the key virulence factors of Cryptococcus is its urease. It plays a crucial role in the ability of Cryptococcus to survive in the host’s respiratory tract and central nervous system (CNS), promoting fungal growth and causing tissue damage. Cryptococcus has been shown to produce significantly higher levels of urease in the CNS compared to other parts of the body, which suggests that urease may play a key role in CNS infections caused by this fungus.²³ The CNS is particularly vulnerable to these infections, which can lead to meningitis and meningoencephalitis. Studies have shown that urease activity in Cryptococcus can directly contribute to CNS damage by increasing the production of ammonia, which can lead to the formation of brain edema and neuronal damage. In addition, urease activity in Cryptococcus may also play a role in the suppression of the host immune response, allowing the fungus to better establish itself within the CNS.²⁴

Urease has been used as a diagnostic tool in various clinical settings. For example, the presence of urease in biopsy specimens can be used to identify bacterial and fungal infections. Urease breath tests are used to diagnose H. pylori infections. The test involves measuring the amount of carbon dioxide in a patient’s breath after they ingest a urea solution. The presence of carbon dioxide indicates the presence of the bacterium.²⁵

Regarding industrial applications, the enzyme is used in the production of fertilizer, where it hydrolyzes urea into ammonia and carbon dioxide, which can be used as a source of nitrogen for plants. Urease is also used in the beverage industry to control urea levels in fermented products.²⁶

Urease has been found to play a vital role in biomineralization. Biomineralization is the process by which living organisms form minerals such as bones and shells. Urease is involved in the formation of calcium carbonate, a substance found in many biominerals.²⁷ The enzyme hydrolyzes urea, releasing ammonia, which then reacts with carbon dioxide to form bicarbonate ions. These ions then react with calcium ions to form solid calcium carbonate. Researchers are exploring the use of urease in biomineralization for various applications. For example, the production of self-healing concrete has been a topic of interest in the field of architecture. Urease can be used to create calcium carbonate in concrete, which can help repair cracks in the material.²⁸

Furthermore, urease has been used as a biocatalyst in industrial applications such as wastewater treatment and bioremediation. The ammonium produced can be used as a nitrogen source for microorganisms involved in these processes. Urease has also been used in the production of biosensors and nanomaterials.²⁶

The inhibition of urease activity is heavily used in agriculture, aiming at free urease in soil that degrades fertilizer ammonia.²⁹ Urease inhibition could be a potential therapeutic strategy for bacterial and fungal infections. By inhibiting urease activity, the production of ammonia is prevented, hindering the survival of these pathogens in the host. Thus developing urease inhibitors could lead to new treatments for these infections.³⁰ Still, being a moonlighting protein, urease shows additional (deleterious) effects that would not be inhibited by traditional catalysis inhibitors, challenging the traditional drug development strategies.¹³

1.4 Conclusions

Urease is a versatile enzyme with numerous biotechnological applications. Its use in the diagnosis of infections and biotechnological and industrial applications highlights its potential in various fields. Targeting urease activity could lead to new therapeutic strategies for bacterial and fungal infections, while additional urease functions are still being investigated and would benefit from putative multitarget inhibitors.

References

1. Krajewska B. Ureases I Functional, catalytic and kinetic properties: a review. J Mol Catal B: Enzymatic. 2009;59(1–3):9–21.

2. Carlini CR, Ligabue-Braun R. Ureases as multifunctional toxic proteins: A review. Toxicon. 2016;110:90–109.

3. Kappaun K, Piovesan AR, Carlini CR, Ligabue-Braun R. Ureases: historical aspects, catalytic, and non-catalytic properties - a review. J Adv Res. 2018;13:3–17.

4. Maroney MJ, Ciurli S. Nonredox nickel enzymes. Chem Rev. 2014;114(8):4206–4228.

5. Tsang KL, Wong KB. Moving nickel along the hydrogenase-urease maturation pathway. Metallomics: Integr biometal Sci. 2022;14(5):mfac003.

6. Ligabue-Braun R, Andreis FC, Verli H, Carlini CR. 3-to-1: unraveling structural transitions in ureases. Die Naturwissenschaften. 2013;100(5):459–467.

7. Holm L, Sander C. An evolutionary treasure: unification of a broad set of amidohydrolases related to urease. Proteins. 1997;28:72–82.

8. Villalobos-Cid M, Dorn M, Contreras Á, Inostroza-Ponta M. An evolutionary algorithm based on parsimony for the multiobjective phylogenetic network inference problem. Appl Soft Comput. 2023;139:110270 110270.

9. Dixon NE, Gazzola TC, Blakeley RL, Zermer B. Jack bean urease (EC 3.5.1.5) A metalloenzyme A simple biological role for nickel?. J Am Chem Soc. 1975;97(14):4131–4133.

10. Sumner JB. The isolation and crystallization of the enzyme urease. J Biol Chem. 1926;69(2):435–441.

11. Zambelli B, Musiani F, Benini S, Ciurli S. Chemistry of Ni2+ in urease: sensing, trafficking, and catalysis. Acc Chem Res. 2011;44(7):520–530.

12. Carter EL, Tronrud DE, Taber SR, Karplus PA, Hausinger RP. Iron-containing urease in a pathogenic bacterium. Proc Natl Acad Sci U S Am. 2011;108(32):13095–13099.

13. Ligabue-Braun R, Carlini CR. Moonlighting toxins: ureases and beyond. In: Gopalakrishnakone P, Carlini C, Ligabue-Braun R, eds. Plant Toxins. Dordrecht: Springer; 2015;.

14. Carter EL, Flugga N, Boer JL, Mulrooney SB, Hausinger RP. Interplay of metal ions and urease. Metallomics: Integr biometal Sci. 2009;1(3):207–221.

15. Nim YS, Fong IYH, Deme J, et al. Delivering a toxic metal to the active site of urease. Sci Adv. 2023;9(16):eadf7790.

16. Mazzei L, Musiani F, Ciurli S. The structure-based reaction mechanism of urease, a nickel dependent enzyme: tale of a long debate. J Biol Inorg Chem. 2020;25(6):829–845.

17. Mora D, Arioli S. Microbial urease in health and disease. PLoS Pathog. 2014;10(12):e1004472.

18. Lima de Souza Gonçalves V, Cordeiro Santos ML, Silva Luz M, et al. From Helicobacter pylori infection to gastric cancer: current evidence on the immune response. World J Clin Oncol. 2022;13(3):186–199.

19. Baj J, Forma A, Sitarz M, et al. Helicobacter pylori virulence factors-mechanisms of bacterial pathogenicity in the gastric microenvironment. Cells. 2020;10(1):27.

20. Armbruster CE, Mobley HLT, Pearson MM. Pathogenesis of Proteus mirabilis infection. EcoSal Plus. 2018;8.

21. Norsworthy AN, Pearson MM. From catheter to kidney stone: the uropathogenic lifestyle of Proteus mirabilis. Trends microbiology. 2017;25(4):304–315.

22. Gushiken AC, Saharia KK, Baddley JW. Cryptococcosis. Infect Dis Clin North Am. 2021;35(2):493–514.

23. Zaragozam O. Basic principles of the virulence of Cryptococcus. Virulence. 2019;10(1):490–501.

24. Chen Y, Shi ZW, Strickland AB, Shi M. Cryptococcus neoformans infection in the central nervous system: the battle between host and pathogen. J fungi. 2022;8(10):1069.

25. Krajewska B. Ureases II Properties and their customizing by enzyme immobilizations: a review. J Mol Catal B: Enzymatic. 2009;59(1–3):22–40.

26. Qin Y, Cabral JMS. Review properties and applications of urease. Biocatal Biotransformation. 2002;20(1):1–14.

27. Krajewska B. Urease-aided calcium carbonate mineralization for engineering applications: a review. J Adv Res. 2017;13:59–67.

28. Alshalif AF, Irwan JM, Othman N, Al-Gheethi AA, Shamsudin S. A systematic review on bio-sequestration of carbon dioxide in bio-concrete systems: a future direction. Eur J Environ Civ Eng 2020;:1–20.

29. Ray A, Nkwonta C, Forrestal P, et al. Current knowledge on urease and nitrification inhibitors technology and their safety. Rev EnvirHealth. 2021;36(4):477–491.

30. Loharch S, Berlicki Ł. Rational development of bacterial ureases inhibitors. Chem Rec. 2022;22(8):e2022000.

Part II

Historical aspects

Outline

2 Historical hallmarks in urease study

3 Genetics of plant urease, the enzyme that keeps surprising us

4 Microbial ureases

2

Historical hallmarks in urease study

Paula Bacaicoa Caruso¹ and Rodrigo Ligabue-Braun²,    ¹Graduate Program in Biosciences (PPGBio), Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil,    ²Department of Pharmacosciences, and Graduate Program in Biosciences (PPGBio), Universidade Federal de Ciências da Saúde de Porto Alegre – UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil

Abstract

In the early 1700s, an animal substance, later named urea, was obtained for the first time from fermented urine. It was the production of urea from ammonium cyanate that marked the beginning of modern organic chemistry. In 1864 several scientists identified the first ureolytic microorganisms. Ten years later, the first ureolytic enzyme was isolated from putrid urine. The enzyme is now known as urease. Massive amounts of the enzyme for testing were only accessible in the 1900s from soybeans, initially, and then from jack bean, which is still a major source of urease. James Sumner’s work in characterizing this enzyme proved for the first time that enzymes are proteins. Urease also provided the first model for the biological role of nickel as a prosthetic group. Although it was the first enzyme to be crystallized, the structure of urease was not solved until 70 years later. The resolution of the structures confirmed the difference between single-chain ureases in eukaryotes and bacteria, which have at least two chains. Ureases have several isoforms and it is possible to observe different characteristics beyond the catalytic function, such as toxicity and virulence. After more than three centuries, ureases still present challenges to resolve and their involvement in multiple infections highlights the urge for new research intended to elucidate the enzyme activation pathway and its unknown targets.

Keywords

Urease; soybean; jack bean; ureolysis; nickel; moonlighting

2.1 Introduction

Urease is an enzyme that acts by hydrolyzing urea into ammonia and carbonic acid (see Chapter 1 for a primer on ureases). Hence, its history is entwined with urea’s research. Likewise, its enzymatic nature is a milestone in biochemistry, as is its nickel requirement for proper function. In addition to that, urease structure determination has revealed many function-associated features, including catalysis-independent ones. In the following sections, a timeline of urease research is outlined (Figs. 2.1–2.3), culminating with some remarks on urease evolution.

Figure 2.1 Timeline of early urea and urease research (1700–1900).

Figure 2.2 Timeline of urease research (1900–95).

Figure 2.3 Timeline of urease research (2001–present).

2.2 From urea to urease: 1700–1900

It is considered that urea was isolated from human urine in 1773 by the French chemist Hilaire Marin Rouelle.¹ Despite his ubiquitous accreditation in the literature, it has been pointed out² that Rouelle was not the true pioneer in urea isolation. This title would better suit Herman Boerhaave (a Dutch botanist, chemist, and physician), whose purer urea preparation was obtained in 1727. These early achievements in urea isolation were followed by an extensive characterization by the French scientists Antoine François Fourcroy and Louis Nicolas Vauquelin, who studied animal chemistry. In 1798 they presented a lengthy analysis of both fresh and putrefied urine and its behavior.³ Among their propositions was the term urea (urée) for this animal substance, along with the identification of ammonia release from fermented urine.

In 1828 Friedrich Wöhler, a German chemist, proposed a method for producing urea from ammonium cyanate.⁴ Taken as the beginning of modern organic chemistry, the Wöhler synthesis is considered the first proof that an organic compound could be obtained from inorganic substances.⁵,⁶

The first ureolytic organism was identified in 1864 by the French botanist Philippe Édouard Léon van Tieghem.⁷ It was named Micrococcus ureae.⁸ This is yet another case of multiple parenthood, since other scientists were also identifying ureolytic microorganisms in the same period (including Louis Pasteur).⁹

In 1874 Frédéric Alphonse Musculus, a French chemist, was able to isolate what was then called a soluble ferment that was able to yield ammonia from putrid urine.¹⁰ This was in fact the first isolation of a ureolytic enzyme. In 1890 Pierre Miquel, a pioneer in the study of aerobiology, proposed to name this enzyme urease.¹¹

2.3 Enzymes are proteins and they can have nickel: 1900–75

Urease, as it was then named, was being obtained from microorganisms. In such preindustrial/prebiotech scenarios, the yields were minimal. The enzyme only became readily accessible in 1909, when Takeuchi and Inone described their method for obtaining massive amounts of urease from soybeans (Glycine max).¹² Seven years later, Mateer and Marshall described how jack beans (Canavalia ensiformis) had up to 15 times as much urease as soybeans.¹³ From this point onwards, jack bean became the source material for urease tests.¹⁴

With urease now readily available, James Batcheller Sumner embarked in the chemical characterization of enzymes. This work led to the discovery of protein crystals that are able to decompose urea into ammonium carbonate, linking enzymatic activity to a protein source, in a demonstration that enzymes were proteins.¹⁵ This discovery motivated the Nobel Prize in Chemistry awarded to Sumner in 1946.¹⁶ It is noteworthy that such groundbreaking discovery was originally met with little to no reaction from the scientific societies.¹⁷ As can be observed in Sumner’s own recollection of his discovery, allegations against his work ranged from scientific illiteracy to xenophobic comments.¹⁷

From Sumner’s groundbreaking discovery to the 1970s, urease prompted (either being directly or indirectly involved in the experiments) major enhancement of enzyme knowledge. These included topics on structure, biochemistry, stability, proficiency, and specificity.¹⁸,¹⁹ In 1975 the promptly available jack bean urease was demonstrated to require nickel for catalysis.²⁰ Analytical difficulties hindered the proper detection of nickel up to the 1970s.²¹ Despite this limitation, at the time nickel was considered biologically irrelevant. Urease provided the first model for a biological role of this metal as a prosthetic group and, besides urease, only eight other enzymes were shown to contain nickel.²² In his personal recollections, Nicholas Dixon muses on how this nickel detection was caused by a technician looking for more than what was called for in an experiment.²³

2.4 Structure-function(s) of urease: 1981–currently

Despite being the first enzyme to be crystalized (see previous section), the structure of urease was only solved 70 years later. This structure was obtained for recombinant Klebsiella aerogenes urease.²⁴ Soon after, a native Sporosarcina pasteurii urease structure was also solved.²⁵ Both bacterial ureases had the trimeric structure for the functional monomer, which in turn formed a trimer in its native form (trimer of trimers). These initial structures were fundamental for proposing catalysis mechanisms, fueling decades-long debates. For an in-depth coverage of the urease catalysis mechanism proposal through time, see the study by Mazzei et al.¹⁹

The peculiar di-chain urease functional monomer of Helicobacter pylori had its structure solved in 2001,²⁶ revealing a dodecameric supramolecular arrangement (four trimers-of-dimers) (for detailed structural representations, see Chapter 1). In 2008 a similar urease from Helicobacter mustelae was shown to possess iron instead of nickel in its active site.²⁷,²⁸ This was yet another oddity for ureases, especially considering the dodecameric structure of Helicobacter ureases.

In 2010 more than 80 years after its original crystallization,¹⁵ jack bean urease had its structure solved.²⁹ It was soon followed by another legume urease, the one from pigeon pea.³⁰ These plant ureases confirmed the single chain of ureases expected for eukaryotes, encompassing the three (or two) chains previously described in bacteria as domains fused by linker regions. The supramolecular arrangement observed was also different, being either trimeric or hexameric (dimer of trimers). So far, no fungal urease had its structure solved.

In parallel with these discoveries, in 1981 Carlini and Guimarães³¹ described a novel toxin found in Canavalia ensiformis seeds, which they named canatoxin. This toxin was shown to cause seizures and death in mice and rats when injected but not when given orally, and had insecticidal activity against beetles and bugs (but not caterpillars). These observations led to the discovery of a digestion-dependent route of toxicity.³² Twenty years after its original description, canatoxin was revealed to be a urease,³³ with zinc and nickel in its active site, and that jack bean had at least two urease isoforms.³⁴ The current model for urease toxicity supports three ureases in this legume,³⁵ as is shown for soybeans.³⁶

In both jack beans and soybeans, at least one of the urease isoforms is strongly correlated with toxicity, most likely related to defense against herbivory. In soybean, the embryo-specific urease fulfills a defense role in seeds, while the ubiquitous urease performs the canonical physiological role in the nitrogen cycle. The third urease has an incomplete active site domain and is proposed to act solely as a toxin. For jack bean ureases, all three isoforms (JBU, JBU-II, and canatoxin) were shown to be toxic. It is interesting to point out that James Summner made the following observation in 1937: "The jackbean is said to grow wild in Central Africa. It has been imported into the Americas and is grown in the West Indies, the Southern States and in the Hawaiian Islands. It is said to be used as a fetish by the negroes (sic) to protect their crops."¹⁷ In light of the multiple toxic functions performed by ureases, this might be a misinterpretation of proper biopesticide use of urease in the form of jack bean fencing of other legumes.

The multiple effects of ureases (see chapter 10), including secretion-inducing, insecticidal, and fungicidal/fungitoxic activities, most of them independent of ureolysis, allowed for the classification of ureases as moonlighting proteins.³⁷,³⁸ The participation of ureases in multiple infections (see chapters 4, 11, 12) highlights their relevance beyond catalysis, which in itself can be harmful/toxic by ammonia release.

Despite the varied list of potential urease activities still to be tested (or discovered by pure chance), the current frontier in urease research can be considered to be the enzyme’s activation pathway. Ureases require a set of accessory proteins for proper assembly of the active site, varying from three for plants and four for bacteria (see Chapter 1). Individual roles for each of the accessory proteins have been solved via multiple techniques, highlighting computational modeling and structural determination.

The identity of these proteins in bacteria,³⁹ the requirement for carbon dioxide and GTP for proper activation,⁴⁰,⁴¹ the plant and fungal activation pathways,⁴²–⁴⁴ are all relatively new developments in the study of ureases. One of the accessory proteins, UreG, is also a milestone in biochemistry, being the first intrinsically disordered enzyme to be described.⁴⁵–⁴⁷

The order of binding and stoichiometry of the activation complex(es) are also intensively studied, leaning heavily on the combination of in vitro, in vivo, and in silico observations.⁴⁸–⁵⁶ The amount of research in this area is reflected in the need for frequent reviews covering the urease activation pathway.⁵⁷–⁵⁹

2.5 Urease origins: notes on urease prehistory

The toxicity of ureases bears on its peculiar structure, which is much larger than expected for proteins in the same family. Other amidohydrolases share the catalytic region (α1) that forms the larger α domain in ureases but lack its smaller (α2) subdomain.²⁹ Dihydroorotase is considered ancestral to all amidohydrolases, being, as expected, structurally similar to the α2 subdomain⁵⁰,⁶⁰ (Fig. 2.4). Ureases, however, are considered ancient enzymes. They have been enrolled in the primordial peptide cycle, being responsible for the metal-dependent degradation of urea.⁶¹ In this sense, they would be expected to function as small scaffolds for metal clusters.

Figure 2.4 Comparison of urease and dihydroorotase, highlighting the structural similarity between the latter and the catalytic region of the former.

If ureolysis can be totally traced to the urease active site, it becomes apparent that there is a surplus of protein structure in regards to this activity.³⁵ The noncatalytic regions of ureases have been associated with proper gating of nickel ions and are gradually being mapped for potential toxicity. The evolutionary requirement of such a large enzyme, with a complex activation mechanism, is still a mystery. This is especially intriguing when one considers the overall streamlining tendency observed for microbial genomes.

Ancestral ureases may have not been enzymes at all. There is the hypothesis that they were originally toxins that acquired a catalytic metallocenter by gene fusion (or transfer), becoming enzymatic. Despite seeming farfetched, this proposition would justify the enormous structure observed for ureases and its accessory proteins.³⁵ If one considers ammonia release as yet another way of causing toxicity, it is possible (even if not confirmable) that ureases evolved as Swiss Army knives, conserving structural regions that increase their menu of (evolutionarily advantageous) toxicity.

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