Assembly of protein metallocenters is not well understood. Urease offers a tractable system for examination of this process. Formation of the urease metallocenter in vivo is known to require four accessory proteins: UreD, postulated to be a urease-specific molecular chaperone; UreE, a nickel(II)-binding protein; and UreF and UreG, of unknown function. Activation of purified Klebsiella aerogenes urease apoprotein was accomplished in vitro by providing carbon dioxide (half-maximal activation at approximately 0.2 percent carbon dioxide) in addition to nickel ion. Activation coincided with carbon dioxide incorporation into urease in a pH-dependent reaction (pKa > or = 9, where Ka is the acid constant). The concentration of carbon dioxide also affected the amount of activation of UreD-urease apoprotein complexes. These results suggest that carbon dioxide binding to urease apoprotein generates a ligand that facilitates productive nickel binding.
The formation of active urease in Kiebsiella aerogenes requires the presence ofthree structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickelcontaining enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid contining the K. aerogenes urease gene cluster or derivatives ofthis plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease.
Klebsiella aerogenes urease in a Ni-containing enzyme (two Ni per alpha beta gamma unit) that is purified as an apoprotein from cells grown in Ni-free medium. Partial activation of urease and UreD-urease apoproteins is achieved in vitro by incubation in the presence of Ni(II) and CO2, whereas incubation of these proteins with Ni alone leads to the formation of inactive species [Park, I.-S., & Hausinger, R. P. (1995) Science 267, 1156-1158]. Here we determined the kinetics of these inhibitory reactions and demonstrated the presence of two Ni ions per alpha beta gamma unit in the inactive proteins. Although metal-substituted urease has never been purified from Ni-deprived cell, several other metal ions were shown to bind to the urease apoproteins. Divalent Zn, C, Co, and Mn all inhibited Ni- and Co2-promoted urease activation at concentrations below that of Ni, whereas Mg and Ca ions did not inhibit this process. Ni-inhibited species recovered their ability to be partially activated after EDTA treatment. In contrast, samples that were exposed to Co or Cu ions were irreversibly inactivated, and EDTA treatment of Zn- or Mn-inhibited samples led to reduced levels of activation competence. Mn-substituted urease, generated from urease apoprotein samples in a Mn- and Co2-dependent manner, was shown to be active, whereas other metal-substituted forms if urease lacked activity. The Mn-protein possessed only 2% of the activity of Ni-activated apoprotein [ approximately 8.0 vs approximately 400 mumol min-1 (mg protein)-1], but its KM value was only moderately altered from that of the native enzyme (3.86 +/- 0.15 mM vs 0.2 mM). Unlike the Ni-containing enzyme, Mn-urease was inhibited by EDTA. Given the evidence that urease apoprotein binds numerous metal ions, we speculate on possible roles for the UreD, UreF, and UreG accessory proteins in urease activation.
To investigate the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin (IN), a series of IN analogs with Trp-->Lys substitution were synthesized. All IN analogs displayed an approximately 7- to 18-fold higher cell selectivity, compared with IN. IN, IN-1 and IN-2 depolarized (50-90%) the cytoplasmic membrane potential of Staphylococcus aureus close to minimal inhibitory concentration (5-10 microg mL(-1)). However, other IN analogs (IN-3 and IN-4) displayed very low ability in membrane depolarization even at 40 microg mL(-1). Confocal laser-scanning microscopy revealed that IN-3 and IN-4 penetrated the Escherichia coli cell membrane, whereas IN, IN-1 and IN-2 did not enter the cell membrane. In the gel retardation assay, IN-3 and IN-4 bound more strongly to DNA compared with IN, IN-1 and IN-2. These findings suggest that the mechanism of antimicrobial action of IN-3 and IN-4 may be involved in the inhibition of intracellular functions via interference with DNA/RNA synthesis. Unlike IN, all IN analogs did not inhibit nitric oxide production or inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells, indicating that the hydrophobicity of IN is more important for anti-inflammatory activity in lipopolysaccharide-treated macrophage cells than the positive charge.
A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R. P. (1993) Protein Sci. 2, 1034 -1041). The crystal structure of the H134A protein was obtained at 2.0-Å resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present. In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules. The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies. The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors. These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry.Urease (EC 3.5.1.5) is a nickel-containing enzyme that catalyzes the hydrolysis of urea (1, 2). In addition to playing a key role in plant (3) and microbial (4) nitrogen metabolism, the enzyme has been implicated as a virulence factor in various human and animal pathogens (reviewed in Ref.2). The threedimensional structure has been resolved to 2.2 Å (5) for the best characterized urease, that from the enteric bacterium Klebsiella aerogenes. The protein is a trimer of trimers ((␣␥) 3 ) composed of subunits with M r ϭ 60,304 (␣), 11,695 (), and 11,086 (␥). The enzyme active site is located in the ␣-subunit and contains a binickel center in which the two metal ions are separated by 3.5 Å and bridged by carbamylated Lys-217. In the crystallographic model, one nickel ion (Ni-2) exhibits distorted trigonal bipyramidal or distorted square pyramidal geometry in which two nitrogen ligands are derived from His-134 and His-136, and three oxygen atoms are contributed by carbamylated Lys-217, Asp-360, and a solvent molecule (Wat-1). 1The second urease metal ion (Ni-1) exhibits pseudotetrahedral geometry, a coordination that is unusual for nickel. The ligands to Ni-1 include the second oxygen atom of the carbamylated Lys-217, nitrogen atoms from His-246 and His-272, and partial coordination to the solvent molecule that is strongly coordinated to Ni-2.In an effort to better characterize the novel coordination geometry observed in the Ni-1 site of urease, we have examined a mutant with His-134 substituted by Ala (H134A) that contains only this metal ion (6). We compare the crystal structure and the UV-visible, XAS, and VTMCD spectroscopic signatures of the H134A protein to the corresponding properties of the native enzyme (5, 7-9).2 We demonstrate that the Ni-1 metallo...
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