Chapter 16 : Urease

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 ( 46 ). Lanes (from left to right): crude lysate of H. pylori, 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.

Space-filling model of the predicted urease crystal structure. The primary amino acid sequence of H. pylori urease was overlaid onto the solved crystal structure of Klebsiella aerogenes urease ( 49 , 50 ). 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 H. pylori urease has not been determined directly (figure designed by Ron Guiles and Nereus Gunther, University of Maryland).

Model for synthesis of a catalytically active urease in H. pylori. The urease gene cluster, composed of seven chromosomally encoded genes, is present as a single copy on the chromosome. The genes ureA and ureB 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 Klebsiella aerogenes urease ( 49 ). The known molecular size of H. pylori urease (550 kDa) would require two of the depicted protein structures to be associated in some manner ( 46 ). This arrangement has not yet been solved. Accessory genes ureE, ureF, ureG, and ureH encode accessory proteins UreE, UreF, UreG, and UreH, which, by analogy to homologs of other species, serve to insert nickel ions (Ni2+) into the apoenzyme in an energy-requiring reaction ( 71 ). UreE is a nickel-binding dimer. UreG carries a GTP-binding site. The gene ureI 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 ( 101 ). Two nickel ions are coordinated into the active site of each UreB subunit. Thus, each H. pylori urease contains 12 nickel ions when fully activated. Nickel ions are transported into the cell by NixA, a high-affinity membrane transport protein ( 70 ). 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.)

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 ( 34 ). 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 34 .)