Chapter 16 : Urease

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Urease is produced by numerous taxonomically diverse bacterial species, including normal flora and nonpathogens. This enzyme is used for taxonomic identification and for diagnosis and follow-up after treatment, and is a vaccine candidate. synthesizes an extraordinary amount of urease. The purified enzyme, however, is not significantly more active than purified ureases from other species, but it simply represents a larger proportion of total cell protein in this species. Purified native urease has been examined by transmission electron microscopy and appears as a round, doughnut-shaped, hexagonal particle with a darkly staining core. The crystal structure of the related urease from has been solved by X-ray diffraction. For synthesis of a catalytically active urease, the accessory genes , , , , and also must be expressed. Mutants that lacked detectable urease activity were readily selected and had no apparent alteration of growth rates, demonstrating that enzyme activity was not necessary for viability in vitro. Polymerase chain reaction (PCR) amplification of urease genes has been used in methods to establish the presence of viable or nonviable . Studies in monkeys with purified urease apoenzyme or in humans using salmonella / deletion mutant expressing the apoenzyme demonstrated little or no protection against infection. Other studies in monkeys, however, showed significant reduction in colonization.

Citation: Mobley H. 2001. Urease, p 179-191. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch16
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Figure 1

Purified urease electrophoresed on an SDS-polyacrylamide gel. Protein (10 µg) from each purification step was electrophoresed on a 10 to 20% polyacrylamide gradient gel and stained with Coomassie Blue ( ). Lanes (from left to right): crude lysate of DEAE-Sepharose, phenyl-Sepharose, Mono-Q, Superose 6, high molecular weight protein standards, low molecular weight protein standards. Molecular masses of the two subunits, predicted from the nucleotide sequence, are shown in the right margin.

Citation: Mobley H. 2001. Urease, p 179-191. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch16
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Figure 2

Space-filling model of the predicted urease crystal structure. The primary amino acid sequence of urease was overlaid onto the solved crystal structure of urease ( ). The front (A) and back (B) views of the two subunits, UreA (dark) and UreB (light) are shown. Note the two nickel atoms inserted into the enzyme active site (panel B). The holoenzyme is composed of six copies of the heterodimer displayed in the figure; the crystal structure of the urease has not been determined directly (figure designed by Ron Guiles and Nereus Gunther, University of Maryland).

Citation: Mobley H. 2001. Urease, p 179-191. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch16
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Figure 3

Model for synthesis of a catalytically active urease in The urease gene cluster, composed of seven chromosomally encoded genes, is present as a single copy on the chromosome. The genes and encode the 26.5-kDa and 60.3-kDa subunits, respectively. Six copies of each subunit spontaneously self-assemble to form the catalytically inactive apoenzyme. The urease protein depicted shows three copies of each subunit and is adapted from the crystal structure of the urease ( ). The known molecular size of urease (550 kDa) would require two of the depicted protein structures to be associated in some manner ( ). This arrangement has not yet been solved. Accessory genes and encode accessory proteins UreE, UreF, UreG, and UreH, which, by analogy to homologs of other species, serve to insert nickel ions (Ni) into the apoenzyme in an energy-requiring reaction ( ). UreE is a nickel-binding dimer. UreG carries a GTP-binding site. The gene is proposed to encode a urea-specific pore in the inner membrane that opens at low pH to allow passage of urea and closes at high pH to prevent access of the substrate to cytoplasmic urease ( ). Two nickel ions are coordinated into the active site of each UreB subunit. Thus, each urease contains 12 nickel ions when fully activated. Nickel ions are transported into the cell by NixA, a high-affinity membrane transport protein ( ). Additional backup nickel transport proteins are also likely present. The net result of the interaction of these genes and proteins is a catalytically active urease. (Figure designed by David McGee.)

Citation: Mobley H. 2001. Urease, p 179-191. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch16
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Figure 4

Topological model of NixA in the cytoplasmic membrane. The amino acid sequence of NixA is presented in single-letter code. Boxed regions indicate transmembrane domains. Filled black diamonds indicate the location of PhoA and LacZ reporter fusions by number (from amino terminus) of the last NixA amino acid residue prior to the fusion junction; enzymatic activity of reporter fusions were used to predict the topology ( ). Circled residues indicate conserved motifs (among known nickel transporters) in helices II and III, plus six additional transport-critical residues. (Reprinted with permission from reference .)

Citation: Mobley H. 2001. Urease, p 179-191. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch16
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