Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0519030 (Klebsiella)
 21,988 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the gamma subunit, H39 and H41 in beta, and H134, H136, H219, H246, H312, H320, and H321 in the alpha subunit of the Klebsiella aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site-directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The gamma H96A, beta H39A, beta H41A, alpha H312A, and alpha H321A mutant proteins possess activities and nickel contents similar to wild-type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the alpha H134A, alpha H136A, and alpha H246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild-type enzyme. These results are consistent with alpha H134, alpha H136, and alpha H246 functioning as nickel ligands. The alpha H219A protein is active and has nickel (approximately 1.9% and approximately 80%, respectively, when compared to wild-type protein) but exhibits a very high Km value (1,100 +/- 40 mM compared to 2.3 +/- 0.2 mM for the wild-type enzyme). These results are compatible with alpha H219 having some role in facilitating substrate binding. Finally, the alpha H320A protein (Km = 8.3 +/- 0.2 mM) only displays approximately 0.003% of the wild-type enzyme activity, despite having a normal nickel content.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis of Klebsiella aerogenes urease: identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis. 831 88

Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163.
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PMID:Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues. 841 30

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-A 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.
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PMID:Characterization of the mononickel metallocenter in H134A mutant urease. 870 15

Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 or Klebsiella pneumoniae has been purified to give three catalytically active fractions. In this mutant, each of the two bridging cysteine ligands to the P-clusters, alpha-Cys-89 and beta-Cys-94, has been replaced by a non-coordinating residue, alanine. SDS/PAGE and earlier native gels showed that the three fractions retained the normal alpha 2 beta 2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chronatography. Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure. The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity. No change in substrate specificity or in the relative distribution of electrons to various substrates was found. The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron-molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1. The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres. This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e. that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein. This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover. We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters. On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco; (ii) the EPR and catalytic inactivation of some FeMoco; and/or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules.
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PMID:Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine. 876 59

The CitA citrate transporter in Salmonella typhimurium is encoded by the citA gene and consists of 434 amino acid residues that probably include 12 membrane-spanning segments [Shimamoto. T., et al. (1991) J. Biochem. 110, 22-28]. CitA mutants with altered substrate specificities were isolated by in vitro mutagenesis using nitrous acid. The mutants could grow on isocitrate as a sole carbon source which normally cannot be transported well by the CitA transporter of S. typhimurium. The mutation sites in the citA gene of the nine mutants were determined to involve single residues at seven sites (one mutation per mutant). The original amino acid residues at these sites (Arg-19, Ala-38, Glu-51, Gly-132, Ala-169, Pro-262 and Leu-271) were identified to be responsible for the altered substrate specificity. All these amino acid residues were conserved in four other homologous citrate transporters from Escherichia coli, Citrobacter amalonaticus and Klebsiella pneumoniae and are suggested to be involved in substrate recognition by the CitA transporter.
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PMID:Mutational analysis of the CitA citrate transporter from Salmonella typhimurium: altered substrate specificity. 880 60

In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE. In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results. Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo. These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell. An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes. The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP. In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin. These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly.
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PMID:Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease. 920 19

SHV-6 was previously identified by its susceptibility pattern and biochemical criteria in a clinical isolate of Klebsiella pneumoniae which was resistant to ceftazidime. It contains only a single point difference with the beta laSHV-1 gene as determined by PCR amplification and nucleotide sequencing. This is the result of a single amino acid substitution, Ala for Asp, at position 179. Directed mutagenesis experiments have shown this substitution to confer selective resistance to ceftazidime in the TEM family.
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PMID:Substitution of alanine for aspartate at position 179 in the SHV-6 extended-spectrum beta-lactamase. 922 83

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).
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PMID:Variability of chromosomally encoded beta-lactamases from Klebsiella oxytoca. 925 34

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.
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PMID:Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein. 945 58

Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.
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PMID:Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand. 955 61


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