Urease

Urease (EC 3.5.1.5) belongs to hydrolases, is a member of amidohydrolases and phosphotriesterases superfamily. The systematic name of urease is urea amidohydrolase, a nickel-containing enzyme. Urease is a metalloenzymes and was found in plant, fungi, bacteria, but not in animal. Urease carries two Ni²⁺ ions in their active sites and catalyzes the hydrolysis of urea to produce ammonia and carbamate. Urease contributed two important historical significance in biochemistry. First, the crystallization of urease demonstrated the proteinaceous nature of enzymes. Second, the biological significance of nickel was recognized.

Structure

X-ray crystallography studies have shown that both plant and bacteria ureases share a common trimeric structure, but the number of polypeptide chains that form monomers or functional units is different. The functional unit of plant and fungal ureases is a single polypeptide chain (α), while the functional unit of bacterial ureases is formed by two (α and β, only found in Helicobacter) or three (α, β and γ) polypeptide chains. The most abundant structure of plant ureases is a dimer of trimers (α₃)₂, a few plant and fungal ureases are dimeric/trimeric/tetrameric. Bacterial ureases are trimers (αβγ)3, Helicobacter pylori’s urease is a a tetramer of trimers of dimers ([αβ]₃)₄. The amino acid sequence of the smaller subunit of the pronuclear urease is collinear with the corresponding region in the single chain of the eukaryotic urease.

Figure 1. Structure of ureases. (Kappaun K. 2018)

Catalytic Mechanism

The active site of ureases consists of two nickel atoms, one carbamylated lysine, four histidines and one aspartate residue. The carbamylated lysine bridge connects two nickel atoms, Ni(1) coordinated by two histidines, and Ni(2) coordinated by the other two histidines and one aspartate residue. The hydroxide ion bridge connects the two Ni atoms, together with the other three terminal water molecules, forms a hydrogen-bonded water tetrahedral cluster in the active site. During its catalytic reaction, urea replaces the three water molecules at the active site first, and then combines with the Ni(1) through its carbonyl oxygen, making the urea carbon more electrophilic and more susceptible to nucleophilic attack. Urea then combines with Ni(2) through its amino nitrogen atom to form a bidentate bond with ureases. This combination promotes nucleophilic attack of the carbonyl carbon by the water molecule to form a tetrahedral intermediate from which NH₃ and carbamate are released.

Figure 2. Catalytic mechanism of ureases. (Kappaun K. 2018)

urease activation

In order to achieve ureolytic activity, the active site of the urease needs to be inserted with nickel ions and also requires carbamoylation of its lysine residue. In bacteria, four accessory proteins (UreD, UreF, UreG and UreE) are involved in the assembly of urease active metal centers. Urease activation starts with UreD, the first protein bind to apo-urease oligomers as a scaffold for the formation of the activation complex. UreF then binds to (UreABC–UreD)₃ and acts as a GTPase activating protein. Finally, the combination of UreG completes the formation of the complex. UreG is an intrinsically disordered enzyme that acts as a GTPase to delivery energy for the urease maturation process. When GTP is hydrolyzed, the nickel binding chaperone UreE delivers metal ions to (UreABC–UreD)₃. This activation model has been further improved with the increase in structural information of the urease accessory protein. In the new activation model, UreE combined with Ni²⁺ binds apo-UreG to promote GTP uptake by UreG, and Ni²⁺ is translocated from UreE to UreG. The (UreDF)₂ complex then competes with UreE for Ni²⁺-UreG to form a supercomplex apo-urease/Ni²⁺-(UreDFG)₂. Finally, KHCO₃/NH₄HCO₃ catalyzes the hydrolysis of GTP by UreG to complete the activation of urease.

Ureases inhibitors

There are two main purposes for studying ureases inhibitors. One is to understand how catalytic sites work, and the other is to find effective inhibitors that counterbalance ureases’ catalyzed urea hydrolysis in certain situations. Ureases have a variety of inhibitors, including sulfur compounds, hydroxamines, phosphorous compounds, fluoride, quinones and polyphenols. Thiols inhibit ureases in a competitive manner. Acetohydroxamic acid is a slow-binding competitive inhibitor of ureases which can be used to reduce urinary stones and treat urinary infections. Derivatives of phosphoric acid and phosphorothioate are potent inhibitors of urease. Phosphate is a pH-dependent urease competitive inhibitor with a pH range of 5.0-8.0, but negligible at pH values above 7.5-8.0. Fluoride is also a PH sensitive inhibitor. Studies on quinones demonstrated a general slow-binding concentration-dependent mechanism. Quinones inhibit ureases by covalent modification of the conserved cysteine residue in the active site, also can by arylation and oxidation of its thiol groups.

Applications

Urease enables microorganisms to use urea as their sole source of nitrogen. Urease synthesis is constitutive or synthesized to be a bacterial stress-related response to counteract low environmental pH. The urease of the human gut microbiota hydrolyzes up to 30% of all urea produced in our body. Microbial urease is also important in dental health. The alkali produced by salivary urea cleavage by oral microbial urease can inhibit the formation of cavities and plaque. In ruminants, animal-derived urea is cleaved by bacterial urease in the anterior stomach, releasing ammonia as a nitrogen source for the rumen microbiota, which in turn feeds the animal as biomass.

Reference

1. Kappaun K., Piovesan R.A., Carlini C.R., Braun R.L. Ureases: Historical aspects, catalytic, and non-catalytic properties. Journal of Advanced Research, 2018, 13: 3-17.