Biomedical subjects

R P Hausinger

Publications and source records attributed to R P Hausinger.

At least 19 recordsLinked to original sources

PubMed1992

Preliminary crystallographic studies of urease from jack bean and from Klebsiella aerogenes.

Ureases from both jack bean (Canavalia ensiformis) seeds and Klebsiella aerogenes have been crystallized by the hanging drop method. The plant-derived urease crystals are regular octahedra analogous to those obtained by Sumner. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group F4(1)32, with a = 364 A, and appear to contain one or two subunits in the asymmetric unit. Using a synchrotron source, the crystals diffract to near 3.5 A resolution. Crystals of urease from K. aerogenes belong to the cubic space group I23 or I2(1)3, with a = 170.8 A and appear to contain a single catalytic unit per asymmetric unit. The crystals diffract to better than 2.0 A resolution and are well suited for structural analysis.

Bacterial Proteins↗

PubMed1992

Site-directed mutagenesis of the active site cysteine in Klebsiella aerogenes urease.

Cysteine 319 in the large subunit of Klebsiella aerogenes urease was identified as an essential catalytic residue based on chemical modification studies (Todd, M.J., and Hausinger, R.P. (1991) J. Biol. Chem. 266, 24327-24331). Through site-directed mutagenesis, this cysteine has been changed independently to alanine, serine, aspartate, and tyrosine. None of these mutations (C319A, C319S, C319D, and C319Y, respectively) affected the size or level of synthesis of the urease subunits as monitored by polyacrylamide gel electrophoresis. The wild type enzyme and each of the mutant proteins was purified and their properties were compared. The C319Y protein possessed no detectable activity, while activity was reduced in C319A, C319S, and C319D to 48, 4.5, and 0.03% of wild type levels under normal assay conditions. All of the active mutants had a small increase in Km when compared to the wild type value. The active mutants displayed a greatly reduced sensitivity to inactivation by iodoacetamide in comparison to the wild type enzyme, confirming our previous assignment of the essential cysteine to this residue based on active site peptide mapping. In contrast to the wild type enzyme, inactivation of the mutant proteins was not affected by the presence of the competitive inhibitor phosphate, suggesting that the remaining slow rate of iodoacetamide inactivation is due to modification away from the active site. The pH dependence of urease activity was substantially altered in the active mutants with C319S and C319D showing a pH optimum near 5.2, and C319A near 6.7, compared to the pH 7.75 optimum of wild type urease. These data are consistent with Cys-319 facilitating catalysis at neutral and basic pH values by participating as a general acid.

Base Sequence↗

PubMed1992

Characterization of urease from Sporosarcina ureae.

Alkaline stable (pH 7.75-12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably active with a specific activity of greater than 9300 mumol urea degraded min-1 mg protein-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to possess a homopolymeric urease containing 1 nickel per subunit [M(r) = 65000], the S. ureae enzyme is comprised of three subunits [apparent M(r) = 63,100 (alpha), 14,500 (beta), and 8500 (gamma)] in an estimated alpha beta gamma 2 stoichiometry and contains 2.1 +/- 0.6 nickel ions per alpha beta gamma 2 unit as measured by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment, dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells.

Amino Acid Sequence↗

PubMed1992

Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis.

The region located immediately upstream from the Klebsiella aerogenes urease structural genes was sequenced and shown to possess an open reading frame capable of encoding a 29.8-kDa peptide. Deletions were generated in this gene, denoted ureD, and in each of the genes (ureE, ureF, and ureG) located immediately downstream of the three structural genes. Transformation of the mutated plasmids into Escherichia coli resulted in high levels of urease expression, but the enzyme was inactive (deletions in ureD, ureF, or ureG) or only partially active (deletions in ureE). Ureases were purified from the recombinant cells and shown to be identical to control enzyme when analyzed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, in every case the activity levels correlated to nickel contents as analyzed by atomic absorption analysis. UreD, UreE, UreF, and UreG peptides were tentatively identified by gel electrophoretic comparison of mutant and control cell extracts, by in vivo expression of separately cloned genes, or by in vitro transcription-translation analyses; the assignments were confirmed for UreE and UreG by amino-terminal sequencing. The latter peptides (apparent M(r)s, 23,900 and 28,500) were present at high levels comparable to those of the urease subunits, whereas the amounts of UreF (apparent M(r), 27,000) and UreD (apparent M(r), 29,300) were greatly reduced, perhaps because of the lack of good ribosome binding sites in the regions upstream of these open reading frames. These results demonstrate that all four accessory genes are necessary for the functional incorporation of the urease metallocenter.

Amino Acid Sequence↗

PubMed1991

Identification of the essential cysteine residue in Klebsiella aerogenes urease.

During reaction with [14C]iodoacetamide at pH 6.3, radioactivity was incorporated primarily into a single Klebsiella aerogenes urease peptide concomitant with activity loss. This peptide was protected from modification at pH 6.3 by inclusion of phosphate, a competitive inhibitor of urease, which also protected the enzyme from inactivation. At pH 8.5, several peptides were alkylated; however, modification of one peptide, identical to that modified at pH 6.3, paralleled activity loss. The N-terminal amino acid sequence and composition of the peptide containing the essential thiol was determined. Previous enzyme inactivation studies of K. aerogenes urease could not distinguish whether one or two essential thiols were present per active site (Todd, M. J., and Hausinger, R. P. (1991) J. Biol. Chem. 266, 10260-10267); we conclude that there is a single essential thiol present and identify this residue as Cys319 in the large subunit of the heteropolymeric enzyme.

Amino Acid Sequence↗

PubMed1991

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification.

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.

2,2'-Dipyridyl↗

PubMed1990

Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation.

A 4.8-kilobase-pair region of cloned DNA encoding the genes of the Klebsiella aerogenes urease operon has been sequenced. Six closely spaced open reading frames were found: ureA (encoding a peptide of 11.1 kilodaltons [kDa]), ureB (11.7-kDa peptide), ureC (60.3-kDa peptide), ureE (17.6-kDa peptide), ureF (25.2-kDa peptide), and ureG (21.9-kDa peptide). Immediately after the ureG gene is a putative rho-dependent transcription terminator. The three subunits of the nickel-containing enzyme are encoded by ureA, ureB, and ureC based on protein structural studies and sequence homology to jack bean urease. Potential roles for ureE, ureF, and ureG were explored by deleting these accessory genes from the operon. The deletion mutant produced inactive urease, which was partially purified and found to have the same subunit stoichiometry and native size as the active enzyme but which contained no significant levels of nickel. The three accessory genes were able to activate apo-urease in vivo when they were cloned into a compatible expression vector and cotransformed into cells carrying the plasmid containing ureA, ureB, and ureC. Thus, one or more of the ureE, ureF, or ureG gene products are involved in nickel incorporation into urease.

Amino Acid Sequence↗

PubMed1990

Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme.

Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79 degrees C) compared with apoenzyme (Tm = 62 degrees C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free cell cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption.

Apoenzymes↗

PubMed1990

Mechanisms of metal ion incorporation into metalloproteins.

Although the structure and function of protein metallocenters have been extensively characterized, much less is known about their assembly. Here, I describe several general strategies for metallocenter biosynthesis and provide literature precedents for each mechanism. The simplest mechanism involves reversible metal ion binding to amino acid ligands of the apo-protein. In a variation of this mechanism, the apo-protein first must be phosphorylated, carboxylated or otherwise covalently modified in order to create the metal ion binding site. Alternatively, passive metal ion binding may require the presence of an associated compound, such as a nucleotide, carbonate or inorganic sulfide, which is co-incorporated into the protein along with the metal ion. In addition, reversible binding may occur using a pre-formed organometallic cofactor such as a metal-tetrapyrrole. Electron transfer reactions are coupled to biosynthesis of certain metallocenters, i.e. oxidation or reduction of the metallocenter or apo-protein may be required prior to binding, or once bound the metallocenter may be oxidatively trapped in the protein. An effector molecule may bind to apo-protein to open up or stabilize the metallocenter binding site, then after the metallocenter is incorporated the effector molecule dissociates. A transferase or insertase protein first may bind the metallocenter and then incorporate it into the appropriate apo-protein. Finally, metal cofactors may be covalently attached to proteins. Regardless of the metallocenter biosynthetic mechanism, intracellular metal ion concentrations must be sufficient; hence, metal ion transport systems also are briefly discussed.

Animals↗

PubMed1989

Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site.

We examined several compounds for their mechanisms of inhibition with the nickel-containing active site of homogeneous Klebsiella aerogenes urease. Thiolate anions competitively inhibit urease and directly interact with the metallocenter, as shown by the pH dependence of inhibition and by UV-visible absorbance spectroscopic studies. Cysteamine, which possesses a cationic beta-amino group, exhibited a high affinity for urease (Ki = 5 microM), whereas thiolates containing anionic carboxyl groups were uniformly poor inhibitors. Phosphate monoanion competitively inhibits a protonated form of urease with a pKa of less than 5. Both the thiolate and phosphate inhibition results are consistent with charge repulsion by an anionic group in the urease active site. Acetohydroxamic acid (AHA) was shown to be a slow-binding competitive inhibitor of urease. This compound forms an initial E.AHA complex which then undergoes a slow transformation to yield an E.AHA* complex; the overall dissociation constant of AHA is 2.6 microM. Phenylphosphorodiamidate, also shown to be a slow-binding competitive inhibitor, possesses an overall dissociation constant of 94 pM. The tight binding of phenylphosphorodiamidate was exploited to demonstrate the presence of two active sites per enzyme molecule. Urease contains 4 mol of nickel/mol enzyme, hence there are two nickel ions/catalytic unit. Each of the two slow-binding inhibitors are proposed to form complexes in which the inhibitor bridges the two active site nickel ions. The inhibition results obtained for K. aerogenes urease are compared with inhibition studies of other ureases and are interpreted in terms of a model for catalysis proposed for the jack bean enzyme (Dixon, N.E., Riddles, P.W., Gazzola, C., Blakely, R.L., and Zerner, B. (1980) Can. J. Biochem. 58, 1335-1344).

Binding Sites↗

PubMed1989

Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease.

The genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The small size of the DNA encoding urease (3.5 kb), the restriction map, and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme.

Bacterial Proteins↗

PubMed1989

Microbial ureases: significance, regulation, and molecular characterization.

Microbial ureases hydrolyze urea to ammonia and carbon dioxide. Urease activity of an infectious microorganism can contribute to the development of urinary stones, pyelonephritis, gastric ulceration, and other diseases. In contrast to these harmful effects, urease activity of ruminal and gastrointestinal microorganisms can benefit both the microbe and host by recycling (thereby conserving) urea nitrogen. Microbial ureases also play an important role in utilization of environmental nitrogenous compounds and urea-based fertilizers. Urease is a high-molecular-weight, multimeric, nickel-containing enzyme. Its cytoplasmic location requires that urea enter the cell for utilization, and in some species energy-dependent urea uptake systems have been detected. Eucaryotic microorganisms possess a homopolymeric urease, analogous to the well-studied plant enzyme composed of six identical subunits. Gram-positive bacteria may also possess homopolymeric ureases, but the evidence for this is not conclusive. In contrast, ureases from gram-negative bacteria studied thus far clearly possess three distinct subunits with Mrs of 65,000 to 73,000 (alpha), 10,000 to 12,000 (beta), and 8,000 to 10,000 (gamma). Tightly bound nickel is present in all ureases and appears to participate in catalysis. Urease genes have been cloned from several species, and nickel-containing recombinant ureases have been characterized. Three structural genes are transcribed on a single messenger ribonucleic acid and translated in the order gamma, beta, and then alpha. In addition to these genes, several other peptides are encoded in the urease operon of some species. The roles for these other genes are not firmly established, but may involve regulation, urea transport, nickel transport, or nickel processing.

Animals↗

PubMed1988

Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids.

Urease was purified 800-fold and partially characterized from Proteus mirabilis, the predominant microorganism associated with urinary stones. Boric acid is a rapid reversible competitive inhibitor of urease. The pH-dependence of inhibition exhibited pKa values of 6.25 and 9.3, where the latter value is probably due to the inherent pKa of boric acid. Three boronic acids also were shown to inhibit urease competitively.

Boric Acids↗

PubMed1988

Partial purification of goat kidney beta-mannosidase.

1. Goat kidney beta-mannosidase was purified 8500-fold to a specific activity of 65,000 nmol/h per mg of protein with a 6% yield by using multiple steps including cation-exchange and anion-exchange fast protein liquid chromatography. This is the first description of a highly purified preparation from goat tissue; however, it was not homogeneous, as judged by silver-stained SDS/polyacrylamide-gel electrophoresis. 2. The enzyme exhibited microheterogeneity when analysed by isoelectric focusing (pI 5.5-6.5). 3. Purified beta-mannosidase hydrolysed the terminal beta-(1----4)-linkage of oligosaccharides that accumulate in beta-mannosidosis.

Animals↗

PubMed1988

Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae.

Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity.

Chromatography, Gel↗

PubMed1988

Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli.

Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.

Autoradiography↗

PubMed1988

Methanogen factor 390 formation: species distribution, reversibility and effects of non-oxidative cellular stresses.

Factor 390 (F390), an adenylylated or guanylylated derivative of the methanogen coenzyme factor 420 (F420), was previously detected in Methanobacterium thermoautotrophicum cells exposed to air. Of six other methanogenic species that have now been tested, only Methanobacterium formicicum was found to produce F390 upon oxygen exposure. Aerobic conditions led to an immediate cessation of methanogenesis, whereas only 51% of cellular F420 was slowly converted to F390 over 4 h in Mb.formicicum at 37 degrees C. F390 formation is reversible. When oxidized cells were re-introduced into anoxic medium, F390 reverted to F420 prior to recovery of methanogenesis. Anaerobic cultures of Mb.formicicum were subjected to alternative stresses such as exposure to heavy metals, methanogenesis inhibitors and eubacterial alarmone-producing chemicals; however, only oxygen was found to induce F390 formation.

2,4-Dinitrophenol↗

PubMed1987

Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes.

Klebsiella aerogenes urease was purified 1,070-fold with a 25% yield by a simple procedure involving DEAE-Sepharose, phenyl-Sepharose, Mono Q, and Superose 6 chromatographies. The enzyme preparation was comprised of three polypeptides with estimated Mr = 72,000, 11,000, and 9,000 in a alpha 2 beta 4 gamma 4 quaternary structure. The three components remained associated during native gel electrophoresis, Mono Q chromatography, and Superose 6 chromatography despite the presence of thiols, glycols, detergents, and varied buffer conditions. The apparent compositional complexity of K. aerogenes urease contrasts with the simple well-characterized homohexameric structure for jack bean urease (Dixon, N. E., Hinds, J. A., Fihelly, A. K., Gazzola, C., Winzor, D. J., Blakeley, R. L., and Zerner, B. (1980) Can. J. Biochem. 58, 1323-1334); however, heteromeric subunit compositions were also observed for the enzymes from Proteus mirabilis, Sporosarcina ureae, and Selemonomas ruminantium. K. aerogenes urease exhibited a Km for urea of 2.8 +/- 0.6 mM and a Vmax of 2,800 +/- 200 mumol of urea min-1 mg-1 at 37 degrees C in 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid, 5.0 mM EDTA buffer, pH 7.75. The enzyme activity was stable in 1% sodium dodecyl sulfate, 5% Triton X-100, 1 M KCl, and over a pH range from 5 to 10.5, with maximum activity observed at pH 7.75. Two active site groups were defined by their pKa values of 6.55 and 8.85. The amino acid composition of K. aerogenes urease more closely resembled that for the enzyme from Brevibacter ammoniagenes (Nakano, H., Takenishi, S., and Watanabe, Y. (1984) Agric. Biol. Chem. 48, 1495-1502) than those for plant ureases. Atomic absorption analysis was used to establish the presence of 2.1 +/- 0.3 mol of nickel per mol of 72,000-dalton subunit in K. aerogenes urease.

Amino Acids↗