Urease

Template:Short description Template:Infobox enzyme Ureases (Template:EC number), functionally, belong to the superfamily of amidohydrolases and phosphotriesterases.<ref name="pmid9144792">Template:Cite journal</ref> Ureases are found in numerous Bacteria, Archaea, fungi, algae, plants, and some invertebrates. Ureases are nickel-containing metalloenzymes of high molecular weight.<ref name="Krajewska">Template:Cite journal</ref> Ureases are distinct from Urecases are important in degrading avian faecal matter, which is rich in uric acid, the breakdown product of which is urea, which is then degraded by urease as described here.

These enzymes catalyze the hydrolysis of urea into carbon dioxide and ammonia:

(NH₂)₂CO + H₂O Template:Overset CO₂ + 2NH₃

The hydrolysis of urea occurs in two stages. In the first stage, ammonia and carbamic acid are produced. The carbamate spontaneously and rapidly hydrolyzes to ammonia and carbonic acid. Urease activity increases the pH of its environment as ammonia is produced, which is basic.

History

Urease activity was first identified in 1876 by Frédéric Alphonse Musculus as a soluble ferment.<ref>Musculus, « Sur le ferment de l'urée », Comptes rendus de l'Académie des sciences, vol. 82, 1876, pp. 333-336, reachable in Gallica</ref> In 1926, James B. Sumner, showed that urease is a protein by examining its crystallized form.<ref name="karplus">Template:Cite journal</ref> Sumner's work was the first demonstration that a protein can function as an enzyme and led eventually to the recognition that most enzymes are in fact proteins. Urease was the first enzyme crystallized. For this work, Sumner was awarded the Nobel prize in chemistry in 1946.<ref>The Nobel Prize in Chemistry 1946</ref> The crystal structure of urease was first solved by P. A. Karplus in 1995.<ref name="karplus"/>

Importance

Urease is important because of its role in the Nitrogen cycle as a key catalyst in the reaction converting urea to ammonium and CO₂. Urease occurs as a soil enzyme, likely because soil microorganisms benefit from the nitrogen made available by urea degradation in the form of ammonium.<ref>Template:Cite journal</ref>

Structure

A 1984 study focusing on urease from jack bean found that the active site contains a pair of nickel centers.<ref>Template:Cite journal</ref> In vitro activation also has been achieved with manganese and cobalt in place of nickel.<ref name="pmid20046957">Template:Cite journal</ref> Lead salts are inhibiting.

The molecular weight is either 480 kDa or 545 kDa for jack-bean urease (calculated mass from the amino acid sequence). 840 amino acids per molecule, of which 90 are cysteine residues.<ref name="Molecular Catalysis B 2009">Template:Cite journal</ref>

The optimum pH is 7.4 and optimum temperature is 60 °C. Substrates include urea and hydroxyurea.

Bacterial ureases are composed of three distinct subunits, one large catalytic (α 60–76kDa) and two small (β 8–21 kDa, γ 6–14 kDa) commonly forming (αβγ)₃ trimers stoichiometry with a 2-fold symmetric structure (note that the image above gives the structure of the asymmetric unit, one-third of the true biological assembly), they are cysteine-rich enzymes, resulting in the enzyme molar masses between 190 and 300kDa.<ref name="Molecular Catalysis B 2009"/>

An exceptional urease is obtained from Helicobacter sp.. These are composed of two subunits, α(26–31 kDa)-β(61–66 kDa). These subunits form a supramolecular (αβ)₁₂ dodecameric complex.<ref name="pmid11373617">Template:Cite journal</ref> of repeating α-β subunits, each coupled pair of subunits has an active site, for a total of 12 active sites.<ref name="pmid11373617" /> It plays an essential function for survival, neutralizing gastric acid by allowing urea to enter into periplasm via a proton-gated urea channel.<ref name="pmid23222544">Template:Cite journal</ref> The presence of urease is used in the diagnosis of Helicobacter species.

All bacterial ureases are solely cytoplasmic, except for those in Helicobacter pylori, which along with its cytoplasmic activity, has external activity with host cells. In contrast, all plant ureases are cytoplasmic.<ref name="Molecular Catalysis B 2009"/>

Fungal and plant ureases are made up of identical subunits (~90 kDa each), most commonly assembled as trimers and hexamers. For example, jack bean urease has two structural and one catalytic subunit. The α subunit contains the active site, it is composed of 840 amino acids per molecule (90 cysteines), its molecular mass without Ni(II) ions amounting to 90.77 kDa. The mass of the hexamer with the 12 nickel ions is 545.34 kDa. Other examples of homohexameric structures of plant ureases are those of soybean, pigeon pea and cotton seeds enzymes.<ref name="Molecular Catalysis B 2009"/>

It is important to note, that although composed of different types of subunits, ureases from different sources extending from bacteria to plants and fungi exhibit high homology of amino acid sequences. The single plant urease chain is equivalent to a fused γ-β-α organization. The Helicobacter "α" is equivalent to a fusion of the normal bacterial γ-β subunits, while its "β" subunit is equivalent to the normal bacterial α.<ref name="Molecular Catalysis B 2009"/> The three-chain organization is likely ancestral.<ref name="pmid30094078">Template:Cite journal</ref>

Activity

The k_cat/K_m of urease in the processing of urea is 10¹⁴ times greater than the rate of the uncatalyzed elimination reaction of urea.<ref name="karplus"/> There are many reasons for this observation in nature. The proximity of urea to active groups in the active site along with the correct orientation of urea allow hydrolysis to occur rapidly. Urea alone is very stable due to the resonance forms it can adopt. The stability of urea is understood to be due to its resonance energy, which has been estimated at 30–40 kcal/mol.<ref name="karplus"/> This is because the zwitterionic resonance forms all donate electrons to the carbonyl carbon making it less of an electrophile making it less reactive to nucleophilic attack.<ref name="karplus"/>

Active site

The active site of ureases is located in the α (alpha) subunits. It is a bis-μ-hydroxo dimeric nickel center, with an interatomic distance of ~3.5 Å.<ref name="karplus" /> > The Ni(II) pair are weakly antiferromagnetically coupled.<ref>Template:Cite journal</ref> X-ray absorption spectroscopy (XAS) studies of Canavalia ensiformis (jack bean), Klebsiella aerogenes and Sporosarcina pasteurii (formerly known as Bacillus pasteurii)<ref name="Benini, S. 1999"/> confirm 5–6 coordinate nickel ions with exclusively O/N ligation, including two imidazole ligands per nickel.<ref name="pmid20046957" /> Urea substrate is proposed to displace aquo ligands.

Water molecules located towards the opening of the active site form a tetrahedral cluster that fills the cavity site through hydrogen bonds. Some amino acid residues are proposed to form mobile flap of the site, which gate for the substrate.<ref name="Krajewska" /> Cysteine residues are common in the flap region of the enzymes, which have been determined not to be essential in catalysis, although involved in positioning other key residues in the active site appropriately.<ref name="Martin">Template:Cite journal</ref> In Sporosarcina pasteurii urease, the flap was found in the open conformation, while its closed conformation is apparently needed for the reaction.<ref name="Benini, S. 1999">Template:Cite journal</ref>

When compared, the α subunits of Helicobacter pylori urease and other bacterial ureases align with the jack bean ureases.<ref name="Martin" />

The binding of urea to the active site of urease has not been observed.<ref name="Molecular Catalysis B 2009"/>

Proposed mechanisms

Blakeley/Zerner

One mechanism for the catalysis of this reaction by urease was proposed by Blakely and Zerner.<ref name="pmid6788353">Template:Cite journal</ref> It begins with a nucleophilic attack by the carbonyl oxygen of the urea molecule onto the 5-coordinate Ni (Ni-1). A weakly coordinated water ligand is displaced in its place. A lone pair of electrons from one of the nitrogen atoms on the Urea molecule creates a double bond with the central carbon, and the resulting NH₂⁻ of the coordinated substrate interacts with a nearby positively charged group. Blakeley and Zerner proposed this nearby group to be a Carboxylate ion, although deprotonated carboxylates are negatively charged.

A hydroxide ligand on the six coordinate Ni is deprotonated by a base. The carbonyl carbon is subsequently attacked by the electronegative oxygen. A pair of electrons from the nitrogen-carbon double bond returns to the nitrogen and neutralizes the charge on it, while the now 4-coordinate carbon assumes an intermediate tetrahedral orientation.

The breakdown of this intermediate is then helped by a sulfhydryl group of a cysteine located near the active site. A hydrogen bonds to one of the nitrogen atoms, breaking its bond with carbon, and releasing an Template:NH3 molecule. Simultaneously, the bond between the oxygen and the 6-coordinate nickel is broken. This leaves a carbamate ion coordinated to the 5-coordinate Ni, which is then displaced by a water molecule, regenerating the enzyme.

The carbamate produced then spontaneously degrades to produce another ammonia and carbonic acid.<ref name="Zimmer">Template:Cite journal</ref>

Hausinger/Karplus

The mechanism proposed by Hausinger and Karplus attempts to revise some of the issues apparent in the Blakely and Zerner pathway, and focuses on the positions of the side chains making up the urea-binding pocket.<ref name="karplus"/> From the crystal structures from K. aerogenes urease, it was argued that the general base used in the Blakely mechanism, His³²⁰, was too far away from the Ni2-bound water to deprotonate in order to form the attacking hydroxide moiety. In addition, the general acidic ligand required to protonate the urea nitrogen was not identified.<ref name="Jabri">Template:Cite journal</ref> Hausinger and Karplus suggests a reverse protonation scheme, where a protonated form of the His³²⁰ ligand plays the role of the general acid and the Ni2-bound water is already in the deprotonated state.<ref name="karplus"/> The mechanism follows the same path, with the general base omitted (as there is no more need for it) and His³²⁰ donating its proton to form the ammonia molecule, which is then released from the enzyme. While the majority of the His³²⁰ ligands and bound water will not be in their active forms (protonated and deprotonated, respectively,) it was calculated that approximately 0.3% of total urease enzyme would be active at any one time.<ref name="karplus"/> While logically, this would imply that the enzyme is not very efficient, contrary to established knowledge, usage of the reverse protonation scheme provides an advantage in increased reactivity for the active form, balancing out the disadvantage.<ref name="karplus"/> Placing the His³²⁰ ligand as an essential component in the mechanism also takes into account the mobile flap region of the enzyme. As this histidine ligand is part of the mobile flap, binding of the urea substrate for catalysis closes this flap over the active site and with the addition of the hydrogen bonding pattern to urea from other ligands in the pocket, speaks to the selectivity of the urease enzyme for urea.<ref name="karplus"/>

Ciurli/Mangani

The mechanism proposed by Ciurli and Mangani<ref name="pmid21542631">Template:Cite journal</ref> is one of the more recent and currently accepted views of the mechanism of urease and is based primarily on the different roles of the two nickel ions in the active site.<ref name="Benini, S. 1999" /> One of which binds and activates urea, the other nickel ion binds and activates the nucleophilic water molecule.<ref name="Benini, S. 1999"/> With regards to this proposal, urea enters the active site cavity when the mobile ‘flap’ (which allows for the entrance of urea into the active site) is open. Stability of the binding of urea to the active site is achieved via a hydrogen-bonding network, orienting the substrate into the catalytic cavity.<ref name="Benini, S. 1999"/> Urea binds to the five-coordinated nickel (Ni1) with the carbonyl oxygen atom. It approaches the six-coordinated nickel (Ni2) with one of its amino groups and subsequently bridges the two nickel centers.<ref name="Benini, S. 1999"/> The binding of the urea carbonyl oxygen atom to Ni1 is stabilized through the protonation state of His^α222 Nԑ. Additionally, the conformational change from the open to closed state of the mobile flap generates a rearrangement of Ala^α222 carbonyl group in such a way that its oxygen atom points to Ni2.<ref name="Benini, S. 1999"/> The Ala^α170 and Ala^α366 are now oriented in a way that their carbonyl groups act as hydrogen-bond acceptors towards NH₂ group of urea, thus aiding its binding to Ni2.<ref name="Benini, S. 1999"/> Urea is a very poor chelating ligand due to low Lewis base character of its NH₂ groups. However the carbonyl oxygens of Ala^α170 and Ala^α366 enhance the basicity of the NH₂ groups and allow for binding to Ni2.<ref name="Benini, S. 1999"/> Therefore, in this proposed mechanism, the positioning of urea in the active site is induced by the structural features of the active site residues which are positioned to act as hydrogen-bond donors in the vicinity of Ni1 and as acceptors in the vicinity of Ni2.<ref name="Benini, S. 1999"/> The main structural difference between the Ciurli/Mangani mechanism and the other two is that it incorporates a nitrogen, oxygen bridging urea that is attacked by a bridging hydroxide.<ref name=Zimmer />

Action in pathogenesis

Bacterial ureases are often the mode of pathogenesis for many medical conditions. They are associated with hepatic encephalopathy / Hepatic coma, infection stones, and peptic ulceration.<ref name="mobley">Template:Cite journal</ref>

Infection stones

Infection induced urinary stones are a mixture of struvite (MgNH₄PO₄•6H₂O) and carbonate apatite [Ca₁₀(PO₄)6•CO₃].<ref name="mobley"/> These polyvalent ions are soluble but become insoluble when ammonia is produced from microbial urease during urea hydrolysis, as this increases the surrounding environments pH from roughly 6.5 to 9.<ref name="mobley"/> The resultant alkalinization results in stone crystallization.<ref name="mobley"/> In humans the microbial urease, Proteus mirabilis, is the most common in infection induced urinary stones.<ref name="pmid3524996">Template:Cite journal</ref>

Urease in hepatic encephalopathy / hepatic coma

Studies have shown that Helicobacter pylori along with cirrhosis of the liver cause hepatic encephalopathy and hepatic coma.<ref name="agrawal">Template:Cite journal</ref> Helicobacter pylori release microbial ureases into the stomach. The urease hydrolyzes urea to produce ammonia and carbonic acid. As the bacteria are localized to the stomach ammonia produced is readily taken up by the circulatory system from the gastric lumen.<ref name="agrawal"/> This results in elevated ammonia levels in the blood, a condition known as hyperammonemia; eradication of Helicobacter pylori show marked decreases in ammonia levels.<ref name="agrawal"/>

Urease in peptic ulcers

Helicobacter pylori is also the cause of peptic ulcers with its manifestation in 55–68% reported cases.<ref name="tang">Template:Cite journal</ref> This was confirmed by decreased ulcer bleeding and ulcer reoccurrence after eradication of the pathogen.<ref name="tang"/> In the stomach there is an increase in pH of the mucosal lining as a result of urea hydrolysis, which prevents movement of hydrogen ions between gastric glands and gastric lumen.<ref name="mobley"/> In addition, the high ammonia concentrations have an effect on intercellular tight junctions increasing permeability and also disrupting the gastric mucous membrane of the stomach.<ref name="mobley"/><ref>Template:Cite journal</ref>

Occurrence and applications in agriculture

Urea is found naturally in the environment and is also artificially introduced, comprising more than half of all synthetic nitrogen fertilizers used globally.<ref>Template:Cite journal</ref> Heavy use of urea is thought to promote eutrophication, despite the observation that urea is rapidly transformed by microbial ureases, and thus usually does not persist.<ref>Template:Cite journal</ref> Environmental urease activity is often measured as an indicator of the health of microbial communities. In the absence of plants, urease activity in soil is generally attributed to heterotrophic microorganisms, although it has been demonstrated that some chemoautotrophic ammonium oxidizing bacteria are capable of growth on urea as a sole source of carbon, nitrogen, and energy.<ref>Template:Cite journal</ref>

Inhibition in fertilizers

Template:Further The inhibition of urease is a significant goal in agriculture because the rapid breakdown of urea-based fertilizers is wasteful and environmentally damaging.<ref name=Pan>Template:Cite journal</ref> Phenyl phosphorodiamidate and N-(n-butyl)thiophosphoric triamide are two such inhibitors.<ref>Template:Cite journal</ref>

Biomineralization

By promoting the formation of calcium carbonate, ureases are potentially useful for biomineralization-inspired processes.<ref>Template:Cite journal</ref> Notably, microbiologically induced formation of calcium carbonate can be used in making bioconcrete.<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref>

Non-enzymatic action

In addition to acting as an enzyme, some ureases (especially plant ones) have additional effects that persist even when the catalytic function is disabled. These include entomotoxicity, inhibition of fungi, neurotoxicity in mammals, promotion of endocytosis and inflammatory eicosanoid production in mammals, and induction of chemotaxis in bacteria. These activities may be part of a defense mechanism.<ref name="pmid30094078"/>

Urease insect-toxicity was originally noted in canatoxin, an orthologous isoform of jack bean urease. Digestion of the peptide identified a 10-kDa portion most responsible for this effect, termed jaburetox. An analogous portion from the soybean urease is named soyuretox. Studies on insects show that the entire protein is toxic without needing any digestion, however. Nevertheless, the "uretox" peptides, being more concentrated in toxicity, show promise as biopesticides.<ref name="pmid30094078"/>

As diagnostic test

{{#invoke:Labelled list hatnote|labelledList|Main article|Main articles|Main page|Main pages}} Many gastrointestinal or urinary tract pathogens produce urease, enabling the detection of urease to be used as a diagnostic to detect presence of pathogens.

Urease-positive pathogens include:

• Proteus mirabilis and Proteus vulgaris
• Ureaplasma urealyticum, a relative of Mycoplasma spp.
• Nocardia
• Corynebacterium urealyticum
• Cryptococcus spp., an opportunistic fungus
• Helicobacter pylori
• Certain Enteric bacteria including Proteus spp., Klebsiella spp., Morganella, Providencia, and possibly Serratia spp.
• Brucella
• Staphylococcus saprophyticus
• Staphylococcus aureus<ref name="pmid30608981">Template:Cite journal</ref>

Ligands

Inhibitors

A wide range of urease inhibitors of different structural families are known. Inhibition of urease is not only of interest to agriculture, but also to medicine as pathogens like H. pylori produce urease as a survival mechanism. Known structural classes of inhibitors include:<ref>Template:Cite journal</ref><ref>Template:Cite journal</ref>

• Analogues of urea, the strongest being thioureas like 1-(4-chlorophenyl)-3-palmitoylthiourea.
• Phosphoramidates, the most commonly used in agriculture (see above).
• Hydroquinone and quinones. In medicine, the most interesting are quinolones, already a class of widely used antibiotics.
• Some plant metabolites are also urease inhibitors, an example being allicin. These have potential both as environmentally-friendly fertilizer additives<ref>Template:Cite journal</ref> and natural drugs.

Extraction

Template:Missing information First isolated as a crystal in 1926 by Sumner, using acetone solvation and centrifuging.<ref>Template:Cite journal</ref> Modern biochemistry has increased its demand for urease. Jack bean meal,<ref>Template:Cite journal</ref> watermelon seeds,<ref>Template:Cite journal</ref> and pea seeds<ref>Template:Cite journal</ref> have all proven useful sources of urease.

See also

• Urea carboxylase
• Allophanate hydrolase
• Urease test

External links

• Template:Cite book