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53-548: See text Nitrospira (from Latin: nitro, meaning "nitrate" and Greek: spira, meaning "spiral") translate into “a nitrate spiral” is a genus of bacteria within the monophyletic clade of the Nitrospirota phylum. The first member of this genus was described 1986 by Watson et al., isolated from the Gulf of Maine . The bacterium was named Nitrospira marina . Populations were initially thought to be limited to marine ecosystems , but it

106-609: A proton-gated urea channel . 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. 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

159-457: A polyphyletic grouping are not inherited from a common ancestor, but evolved independently. Monophyletic groups are typically characterised by shared derived characteristics ( synapomorphies ), which distinguish organisms in the clade from other organisms. An equivalent term is holophyly . The word "mono-phyly" means "one-tribe" in Greek. These definitions have taken some time to be accepted. When

212-607: A reverse protonation scheme, where a protonated form of the His ligand plays the role of the general acid and the Ni2-bound water is already in the deprotonated state. The mechanism follows the same path, with the general base omitted (as there is no more need for it) and His donating its proton to form the ammonia molecule, which is then released from the enzyme. While the majority of the His ligands and bound water will not be in their active forms (protonated and deprotonated, respectively,) it

265-663: A sole source of carbon, nitrogen, and energy. The inhibition of urease is a significant goal in agriculture because the rapid breakdown of urea-based fertilizers is wasteful and environmentally damaging. Phenyl phosphorodiamidate and N -( n -butyl)thiophosphoric triamide are two such inhibitors. By promoting the formation of calcium carbonate , ureases are potentially useful for biomineralization -inspired processes. Notably, microbiologically induced formation of calcium carbonate can be used in making bioconcrete . In addition to acting as an enzyme, some ureases (especially plant ones) have additional effects that persist even when

318-480: A soluble ferment. In 1926, James B. Sumner , showed that urease is a protein by examining its crystallized form. 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. The crystal structure of urease

371-478: A source of nutrients. Urease encoded within their genome can break urea down to CO 2 and ammonia . The CO 2 can be assimilated by anabolism while the ammonia and organic by-product released by Nitrospira allow ammonium oxidizers and other microbes to co-exist in the same microenvironment . All members of this genus have the nitrite oxidoreductase genes, and thus are all thought to be nitrite-oxidizers. Ever since nitrifying bacteria were discovered it

424-468: 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

477-463: Is a ubiquitous bacterium that plays a role in the nitrogen cycle by performing nitrite oxidation in the second step of nitrification. Nitrospira live in a wide array of environments including but not limited to, drinking water systems, waste treatment plants, rice paddies , forest soils , geothermal springs, and sponge tissue. Despite being abundant in many natural and engineered ecosystems Nitrospira are difficult to culture, so most knowledge of them

530-401: Is also the cause of peptic ulcers with its manifestation in 55–68% reported cases. This was confirmed by decreased ulcer bleeding and ulcer reoccurrence after eradication of the pathogen . 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 . In addition,

583-496: 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. The three-chain organization is likely ancestral. The k cat / K m of urease in the processing of urea is 10 times greater than the rate of the uncatalyzed elimination reaction of urea . There are many reasons for this observation in nature. The proximity of urea to active groups in

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636-490: Is from molecular and genomic data. However, due to their difficulty to be cultivated in laboratory settings, the entire genome was only sequenced in one species, Nitrospira defluvii . In addition, Nitrospira bacteria's 16S rRNA sequences are too dissimilar to use for PCR primers , thus some members go unnoticed. In addition, members of Nitrospira with the capabilities to perform complete nitrification ( comammox bacteria) has also been discovered and cultivated. For

689-506: Is located in the α (alpha) subunits . It is a bis-μ-hydroxo dimeric nickel center, with an interatomic distance of ~3.5 Å. > The Ni(II) pair are weakly antiferromagnetically coupled. X-ray absorption spectroscopy (XAS) studies of Canavalia ensiformis (jack bean), Klebsiella aerogenes and Sporosarcina pasteurii (formerly known as Bacillus pasteurii ) confirm 5–6 coordinate nickel ions with exclusively O/N ligation, including two imidazole ligands per nickel. Urea substrate

742-505: Is produced from microbial urease during urea hydrolysis , as this increases the surrounding environments pH from roughly 6.5 to 9. The resultant alkalinization results in stone crystallization . In humans the microbial urease, Proteus mirabilis , is the most common in infection induced urinary stones. Studies have shown that Helicobacter pylori along with cirrhosis of the liver cause hepatic encephalopathy and hepatic coma . Helicobacter pylori release microbial ureases into

795-467: 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. 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

848-482: The Nitrogen cycle and thus a lot of future studies will be dedicated to it. With these new findings there's now a possibility to mainly use complete nitrification instead of partial nitrification in engineered systems like wastewater treatment plants because complete nitrification results in lower emissions of the greenhouse gases : nitrous oxide and nitric oxide , into the atmosphere. After sequencing and analyzing

901-437: The hydrolysis of urea into carbon dioxide and ammonia : 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. Its activity was first identified in 1876 by Frédéric Alphonse Musculus as

954-420: The outer membrane (6-8 nm thick). The periplasmic space is exceptionally wide (34-41 nm thick), which provides space to accommodate electron-rich molecules. Electron-deprived structures are located in the cytosol and are believed to be glycogen storage vesicles; polyhydroxybutyrate and polyphosphate granules are also identified in the cytoplasm. DNA analysis determined 56.9 +/- 0.4 mol% of

1007-620: The Ciurli/Mangani mechanism and the other two is that it incorporates a nitrogen , oxygen bridging urea that is attacked by a bridging hydroxide . 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. Infection induced urinary stones are a mixture of struvite (MgNH 4 PO 4 •6H 2 O) and carbonate apatite [Ca 10 (PO 4 )6•CO 3 ]. These polyvalent ions are soluble but become insoluble when ammonia

1060-446: The DNA of Nitrospira members, researchers discovered both species had genes encoding ammonia monooxygenase (Amo) and hydroxlyamine dehydrogenase (hao), enzymes that ammonia-oxidizing bacteria (AOB) use to convert ammonia into nitrite. The bacteria possess all necessary sub-units for both enzymes as well as the necessary cell membrane associated proteins and transporters to carry out

1113-772: The DNA to be guanine and cytosine base pairs. Nitrospira are capable of aerobic hydrogen oxidation and nitrite oxidation to obtain electrons, but high concentrations of nitrite have shown to inhibit their growth. The optimal temperature for nitrite oxidation and growth in Nitrospira moscoviensis is 39 °C (can range from 33-44 °C) at a pH range of 7.6-8.0 Despite being commonly classified as obligate chemolithotrophs , some are capable of mixotrophy . For instance, under different environments, Nitrospira can choose to assimilate carbon by carbon fixation or by consuming organic molecules ( glycerol , pyruvate , or formate ). New studies also show that Nitrospira can use urea as

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1166-495: 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. 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. The active site of ureases

1219-408: The active site appropriately. In Sporosarcina pasteurii urease, the flap was found in the open conformation, while its closed conformation is apparently needed for the reaction. When compared, the α subunits of Helicobacter pylori urease and other bacterial ureases align with the jack bean ureases. The binding of urea to the active site of urease has not been observed. One mechanism for

1272-561: 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. 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

1325-419: The binding of urea to the active site is achieved via a hydrogen-bonding network, orienting the substrate into the catalytic cavity. 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. The binding of the urea carbonyl oxygen atom to Ni1 is stabilized through

1378-461: The carbonyl oxygens of Ala and Ala enhance the basicity of the NH 2 groups and allow for binding to Ni2. 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. The main structural difference between

1431-458: The catalysis of this reaction by urease was proposed by Blakely and Zerner. 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 NH 2 of

1484-404: 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. Urease insect-toxicity was originally noted in canatoxin, an orthologous isoform of jack bean urease. Digestion of

1537-404: The cladistics school of thought became mainstream in the 1960s, several alternative definitions were in use. Indeed, taxonomists sometimes used terms without defining them, leading to confusion in the early literature, a confusion which persists. The first diagram shows a phylogenetic tree with two monophyletic groups. The several groups and subgroups are particularly situated as branches of

1590-689: The classification of organisms , monophyly is the condition of a taxonomic grouping being a clade – that is, a grouping of taxa which meets these criteria: Monophyly is contrasted with paraphyly and polyphyly as shown in the second diagram. A paraphyletic grouping meets 1. but not 2., thus consisting of the descendants of a common ancestor, excepting one or more monophyletic subgroups. A polyphyletic grouping meets neither criterion, and instead serves to characterize convergent relationships of biological features rather than genetic relationships – for example, night-active primates, fruit trees, or aquatic insects. As such, these characteristic features of

1643-425: 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

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1696-424: The detection of urease to be used as a diagnostic to detect presence of pathogens. Urease-positive pathogens include: 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: First isolated as

1749-478: The enzyme molar masses between 190 and 300kDa. 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. of repeating α-β subunits, each coupled pair of subunits has an active site, for a total of 12 active sites. It plays an essential function for survival, neutralizing gastric acid by allowing urea to enter into periplasm via

1802-413: The fact that a monophyletic group includes organisms (e.g., genera, species) consisting of all the descendants of a unique common ancestor. Conversely, the term polyphyly , or polyphyletic , builds on the ancient Greek prefix πολύς ( polús ), meaning "many, a lot of", and refers to the fact that a polyphyletic group includes organisms arising from multiple ancestral sources. By comparison,

1855-477: The first step of nitrification. Origins of the Amo gene are debatable as one study found that it is similar to other AOB[3], while another study found the Amo gene to be genetically distinct from other lineages. Current findings indicate that the hao gene is phylogenetically distinct from the hao gene present in other AOB, meaning that they acquired them long ago, likely by horizontal gene transfer . Nitrospira also carry

1908-489: The following description, Nitrospira moscoviensis will be representative of the Nitrospira genus. Nitrospira is a gram-negative nitrite -oxidizing organism with a helical to vibroid morphology (0.9–2.2 × 0.2–0.4 micrometres in size). They are non- planktonic organisms that reside as clumps, known as aggregates, in biofilms . Visualization using transmission electron microscopy (TEM) confirms star-like protrusions on

1961-970: The genes encoding for all the sub-units of nitrite oxidoreductase (nxr), the enzyme that catalyzes the second step of nitrification. The currently accepted taxonomy is based on the List of Prokaryotic names with Standing in Nomenclature (LSPN) and the National Center for Biotechnology Information (NCBI). Phylogeny is based on GTDB 08-RS214 by Genome Taxonomy Database " N. defluvii " Nowka et al. 2015 " N. japonica " Ushiki et al. 2013 " N. lenta " Nowka et al. 2015 N. moscoviensis Ehrich et al. 1995 " Ca. N. inopinata " Daims et al. 2015 " Ca. N. kreftii" Sakoula et al. 2021 " Ca. N. nitrificans" van Kessel et al. 2015 " Ca. N. nitrosa" van Kessel et al. 2015 Species incertae sedis: Monophyly In biological cladistics for

2014-404: The high ammonia concentrations have an effect on intercellular tight junctions increasing permeability and also disrupting the gastric mucous membrane of the stomach. Urea is found naturally in the environment and is also artificially introduced, comprising more than half of all synthetic nitrogen fertilizers used globally. Heavy use of urea is thought to promote eutrophication , despite

2067-402: The mechanism of urease and is based primarily on the different roles of the two nickel ions in the active site. One of which binds and activates urea, the other nickel ion binds and activates the nucleophilic water molecule. 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

2120-433: 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. The mechanism proposed by Ciurli and Mangani is one of the more recent and currently accepted views of

2173-399: 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 NH 3 molecule. Simultaneously, the bond between

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2226-442: The observation that urea is rapidly transformed by microbial ureases, and thus usually does not persist. 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

2279-484: 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 . The mechanism proposed by Hausinger and Karplus attempts to revise some of the issues apparent in the Blakely and Zerner pathway, and focuses on

2332-437: 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 . Many gastrointestinal or urinary tract pathogens produce urease, enabling

2385-436: The positions of the side chains making up the urea-binding pocket. From the crystal structures from K. aerogenes urease, it was argued that the general base used in the Blakely mechanism, His , 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. Hausinger and Karplus suggests

2438-494: The protonation state of His Nԑ. Additionally, the conformational change from the open to closed state of the mobile flap generates a rearrangement of Ala carbonyl group in such a way that its oxygen atom points to Ni2. The Ala and Ala are now oriented in a way that their carbonyl groups act as hydrogen-bond acceptors towards NH 2 group of urea, thus aiding its binding to Ni2. Urea is a very poor chelating ligand due to low Lewis base character of its NH 2 groups. However

2491-421: 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 . This results in elevated ammonia levels in the blood, a condition known as hyperammonemia ; eradication of Helicobacter pylori show marked decreases in ammonia levels. Helicobacter pylori

2544-596: The term paraphyly , or paraphyletic , uses the ancient Greek prefix παρά ( pará ), meaning "beside, near", and refers to the situation in which one or several monophyletic subgroups are left apart from all other descendants of a unique common ancestor. That is, a paraphyletic group is nearly monophyletic, hence the prefix pará . On the broadest scale, definitions fall into two groups. The concepts of monophyly, paraphyly , and polyphyly have been used in deducing key genes for barcoding of diverse group of species. Urease These enzymes catalyze

2597-493: The tree to indicate ordered lineal relationships between all the organisms shown. Further, any group may (or may not) be considered a taxon by modern systematics , depending upon the selection of its members in relation to their common ancestor(s); see second and third diagrams. The term monophyly , or monophyletic , derives from the two Ancient Greek words μόνος ( mónos ), meaning "alone, only, unique", and φῦλον ( phûlon ), meaning "genus, species", and refers to

2650-425: Was accepted that nitrification occurred in two steps, although it would be energetically favourable for one organism to do both steps. Recently Nitrospira members with the abilities to perform complete nitrification ( comammox bacteria) have also been discovered and cultivated as in the case of Nitrospira inopinata . The discovery of commamox organisms within Nitrospira redefine the way bacteria contribute to

2703-436: Was calculated that approximately 0.3% of total urease enzyme would be active at any one time. 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. Placing the His ligand as an essential component in the mechanism also takes into account

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2756-498: Was first solved by P. A. Karplus in 1995. A 1984 study focusing on urease from jack bean found that the active site contains a pair of nickel centers. In vitro activation also has been achieved with manganese and cobalt in place of nickel. 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. The optimum pH

2809-610: Was later discovered to be well-suited for numerous habitats, including activated sludge of wastewater treatment systems, natural biological marine settings (such as the Seine River in France and beaches in Cape Cod in the United States), water circulation biofilters in aquarium tanks, terrestrial systems, fresh and salt water ecosystems, agricultural lands and hot springs . Nitrospira

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