Biomedical subjects

P C Loewen

Publications and source records attributed to P C Loewen.

At least 37 records · Page 2Linked to original sources

PubMed1996

Structure of the heme d of Penicillium vitale and Escherichia coli catalases.

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

Catalase↗

PubMed1995

Comparison of isoniazid oxidation catalyzed by bacterial catalase-peroxidases and horseradish peroxidase.

The physical properties and activities of the purified catalase-peroxidase hydroperoxidase I (HPI) of Escherichia coli (EcHPI) and HPI with a carboxyl-terminal extension of Mycobacterium tuberculosis (MtHPI-e) are compared to those of commercial preparations of horseradish peroxidase (HRP). The catalase-peroxidase proteins had similar absorption spectra and differed primarily in that MtHPI-e has a higher peroxidatic to catalatic activity ratio than EcHPI. Trypsin cleavage of MtHPI-e resulted in the formation of an active catalase-peroxidase lacking the carboxyl-terminal extension. The three enzymes, HRP, MtHPI-e, and EcHPI, mediated the isoniazid- and H2O2-dependent production of radical species, as detected by nitroblue tetrazolium reduction. A constant flux of H2O2, generated in situ from glucose oxidase and glucose was used. MtHPI-e was more effective at isoniazid-dependent radical production than EcHPI and HRP. Similar qualitative results were obtained by staining nondenaturing polyacrylamide gels for activity with nitroblue tetrazolium in the presence of isoniazid and H2O2. The absorbance spectrum of HRP exhibited changes during incubation with isoniazid and H2O2 consistent with the formation of several typical reaction intermediates, whereas the catalase-peroxidases exhibited no distinct spectral changes. The results suggest that the sensitivity of M. tuberculosis to isoniazid may be the result of isoniazid-dependent radical formation by the catalase-peroxidase in the absence of other catalase activities to remove substrate H2O2.

Bacterial Proteins↗

PubMed1995

Template secondary structure can increase the error frequency of the DNA polymerase from Thermus aquaticus.

Amplification of portions of the intergenic spacer between the katE gene and cryptic cel operon of Escherichia coli was accomplished by the polymerase chain reaction using the DNA polymerase from Thermus aquaticus. Nine different segments were amplified and cloned without error, but one 83-bp fragment was amplified with a high error rate such that 32 of 34 selected clones had three or more nucleotide changes from the expected sequence. The changes were all located in two 9-bp segments immediately adjacent to the 3'-ends of the two primers. Moving the end points of the primers to increase the spacing between them resulted in the isolation of significantly fewer error-containing products. It is proposed that stem-loop structures in the template immediately downstream from the primers interfere with an early stage of elongation and cause misincorporation. This is supported by the observation that destabilisation of one of the stem-loop structures reduced the frequency of errors.

Artifacts↗

PubMed1995

Crystal structure of catalase HPII from Escherichia coli.

BACKGROUND: Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms. It serves, in part, to protect the cell from the toxic effects of small peroxides. Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties. HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, monofunctional catalase containing one cis-heme d prosthetic group per subunit of 753 residues. RESULTS: The crystal structure of catalase HPII from E. coli has been determined to 2.8 A resolution. The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry. In the model built, that includes residues 27-753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained. The crystallographic agreement R-factor is 20.1% for 58,477 reflections in the resolution shell 8.0-2.8 A. CONCLUSIONS: Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.

Amino Acid Sequence↗

PubMed1995

The cysteines of catalase HPII of Escherichia coli, including Cys438 which is blocked, do not have a catalytic role.

Site-directed mutagenesis of the katE gene of Escherichia coli was used to change, individually and in combination, Cys438 and Cys669 to serine in catalase HPII. The Cys438-->Ser mutation caused a 30% reduction in the specific activity of the enzyme, whereas the Cys669-->Ser mutation did not affect enzyme activity. The titration of free sulfhydryl groups in HPII revealed that Cys669 was reactive whereas Cys438 was unreactive. Properties of the modification on Cys438 included alkali lability, insensitivity to methylamine, hydroxylamine or reducing agents, and a mass determined by mass spectrometry to be approximately 43 +/- 2 Da. A hemithioacetal structure is consistent with these properties. Although free sulfhydryl groups do not play a significant role in the stability or catalytic mechanism of HPII, the sulfhydryl agent 2-mercaptoethanol caused a 50% inactivation of HPII along with an irreversible change in the absorption spectrum of the protein. Other sulfhydryl agents, including dithiothreitol, cysteine and glutathione, and the organic peroxide, t-butylhydroperoxide, which cannot directly access the active site, do not affect HPII activity, but they do cause a small reversible change in the absorption spectrum, possibly by a mechanism involving superoxide.

Base Sequence↗

PubMed1995

DNA repair is more important than catalase for Salmonella virulence in mice.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxify reactive oxygen species, and DNA repair systems which repair damage resulting from oxidative stress. To determine the relative importance of these two potentially protective defense mechanisms against oxidative stress encountered by Salmonella during infection of the host, a Salmonella typhimurium double mutant unable to produce either the HPI or HPII catalase was constructed, and compared with an isogenic recA mutant deficient in DNA repair. The recA mutant was hypersusceptible to H2O2 at low cell densities in vitro, while the catalase mutant was more susceptible to high H2O2 concentrations at high cell densities. The catalase mutant was found to be resistant to macrophages and retained full murine virulence, in contrast to the recA mutant which previously was shown to be macrophage-sensitive and attenuated in mice. These observations suggest that Salmonella is subjected to low concentrations of H2O2 while at relatively low cell density during infection, conditions requiring an intact DNA repair system but not functional catalase activity.

Acatalasia↗

PubMed1994

NADPH binding and control of catalase compound II formation: comparison of bovine, yeast, and Escherichia coli enzymes.

1. NADPH binds to bovine catalase and to yeast catalases A and T, but not to Escherichia coli catalase HPII. The association was demonstrated using chromatography and fluorimetry. Bound NADPH fluoresces in a similar way to NADPH in solution. 2. Bound NADPH protects bovine and yeast catalases against forming inactive peroxide compound II either via endogenous reductant action or by ferrocyanide reduction during catalytic activity in the presence of slowly generated peroxide. 3. Bound NADPH reduces neither compound I nor compound II of catalase. It apparently reacts with an intermediate formed during the decay of compound I to compound II; this postulated intermediate is an immediate precursor of stable compound II either when the latter is formed by endogenous reductants or when ferrocyanide is used. It represents therefore a new type of hydrogen donor that is not included in the original classification of Keilin and Nicholls [Keilin, D. and Nicholls, P. (1958) Biochim. Biophys. Acta 29, 302-307] 4. A model for NADPH action is presented in which concerted reduction of the ferryl iron and of a neighbouring protein free radical is responsible for the observed NADPH effects. The roles of migrant radical species in mammalian and yeast catalases are compared with similar events in metmyoglobin and cytochrome c peroxidase reactions with peroxides.

Animals↗

PubMed1994

Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state.

A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation.

Alanine↗

PubMed1994

The delta (argF-lacZ)205(U169) deletion greatly enhances resistance to hydrogen peroxide in stationary-phase Escherichia coli.

In this study, we demonstrate that a strain bearing the delta (argF-lacZ)205(U169) deletion exhibits a high level of resistance to hydrogen peroxide compared with its undeleted parent. Our initial investigation of the mechanism behind the observed differences in peroxide resistance when parent and mutant strains are compared indicates that the parent strain carries a region near argF that is responsible for the H2O2-sensitive phenotype, which we have named katC. The H2O2 resistance phenotype of the delta katC [delta (argF-lacZ)205(U169)] mutant strain can be duplicated by Tn9 insertion in a specific locus (katC5::Tn9) which maps near argF. The increased H2O2 resistance of the delta katC and katC5::Tn9 mutant strains can be seen only when cells are grown to stationary phase; exponential-phase cells are unaffected by the presence or absence of katC. This H2O2 resistance mechanism requires functional katE and katF genes, which suggests that the mechanism of H2O2 resistance may involve the activity of the stationary-phase-specific catalase HPII. Cloning, DNA sequencing, and analysis of the katC5::Tn9 insertion allele in comparison with its parent allele implicate two insertion elements, IS1B and IS30B, and suggest that their presence sensitizes parent cells to H2O2.

Alleles↗

PubMed1994

The role of the sigma factor sigma S (KatF) in bacterial global regulation.

The protein encoded by katF (also known as nur, appR, csi-2, abrD, and rpoS in various alleles) has been biochemically confirmed to be an alternate sigma transcription factor and renamed sigma S. Its synthesis is controlled transcriptionally and posttranscriptionally by as yet undefined mechanisms that are active well into stationary phase. sigma S controls a regulon of 30 or more genes expressed in response to starvation and during the transition to stationary phase. Proteins in the regulon, many of which have not been characterized, enhance long-term survival in nutrient-deficient medium and have a diverse group of functions including protection against DNA damage, the determination of morphological changes, the mediation of virulence, osmoprotection, and thermotolerance. Differential expression of subfamilies of genes within the regulon is effected by supplementary regulatory factors, working both individually and in combination to modulate activity of different sigma S-dependent promoters.

Bacteria↗

PubMed1993

Catalase HPII of Escherichia coli catalyzes the conversion of protoheme to cis-heme d.

Catalase HPII from aerobically grown Escherichia coli normally contains heme d but cultures grown with poor or no aeration produce HPII containing a mixture of heme d and protoheme IX. The protoheme component of HPII from anaerobically grown cells is converted into heme d during treatment of the purified enzyme with hydrogen peroxide. It is concluded that heme d found in catalase HPII is formed by the cis-hydroxylation of protoheme in a reaction catalyzed by catalase HPII using hydrogen peroxide as a substrate. The distal His128 residue of HPII is absolutely required for the protoheme to heme d conversion. Two mutant enzymes, Ala128 and Asn128, are catalytically inactive and contain only protoheme, which is unaffected by hydrogen peroxide treatment. The Asn201 residue is not an absolute requirement for heme conversion. The mutant enzyme Ala201 contains predominantly heme d and is partially active. However, insertion of a histidyl residue to give the His201 enzyme interferes with the heme conversion reaction. This mutant form is isolated as a protoheme enzyme with limited activity, and a reversible conversion to a heme d-like species occurs in vitro in the presence of continuously generated hydrogen peroxide.

Amino Acid Sequence↗

PubMed1993

Molecular evolutionary analysis based on the amino acid sequence of catalase.

Heme-containing catalase sequences from 20 different organisms representing prokaryotes, fungi, animals, and plants have been compiled for phylogenetic reconstruction. Phylogenies based on distance and parsimony analysis show that fungal and animal catalases can be derived from one ancestor, whereas bacterial catalases fail to form a monophyletic group. Plant catalases appear to form a second class of catalases that arose independently from a possible prokaryotic ancestor.

Amino Acid Sequence↗

PubMed1993

KatF (sigma S) synthesis in Escherichia coli is subject to posttranscriptional regulation.

A transcriptional fusion of katF to the lacZ gene was expressed at increasingly higher levels throughout the exponential phase, but a translational fusion was expressed at low levels during exponential-phase growth and was induced 160-fold during the transition to stationary phase, implicating a posttranscriptional mechanism in the regulation of KatF synthesis. Mutational analyses suggested that the initiation codon of katF is the second ATG in the previously identified open reading frame.

Bacterial Proteins↗

PubMed1992

The alternative sigma factor katF (rpoS) regulates Salmonella virulence.

Nutrient limitation is a critical signal in Salmonella virulence gene regulation. The katF (rpoS) gene mediates the expression of the Salmonella spv plasmid virulence genes during bacterial starvation. A katF Salmonella mutant has increased susceptibility to nutrient deprivation, oxidative stress, acid stress, and DNA damage, conditions which are relevant to the intraphagosomal environment of host macrophages. Moreover, the katF mutant has significantly reduced virulence in mice. katF encodes an alternative sigma factor of RNA polymerase which coordinately regulates Salmonella virulence.

Animals↗

PubMed1992

Identification of heme macrocycle type by near-infrared magnetic circular dichroism spectroscopy at cryogenic temperatures.

The electron paramagnetic resonance (EPR) and near-infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures. For the first time, ligand-to-metal charge-transfer transitions in the near-infrared have been observed for an Fe(III)-chlorine system. It is shown that near-ultraviolet-to-visible region electronic spectra of 'green' hemes such as these are an unreliable indicator of macrocycle type. However, the combined application of EPR and near-infrared MCD spectroscopies clearly distinguishes between the porphyrin-containing nitrimyoglobin and the chlorine-containing hydroperoxidase II.

Circular Dichroism↗

PubMed1991

The active site structure of E. coli HPII catalase. Evidence favoring coordination of a tyrosinate proximal ligand to the chlorin iron.

E. coli produces 2 catalases known as HPI and HPII. While the heme prosthetic group of the HPII catalase has been established to be a dihydroporphyrin or chlorin, the identity of the proximal ligand to the iron has not been addressed. The magnetic circular dichroism (MCD) spectrum of native ferric HPII catalase is very similar to those of a 5-coordinate phenolate-ligated ferric chlorin complex, a model for tyrosinate proximal ligation, as well as of chlorin-reconstituted ferric horseradish peroxidase, a model for 5-coordinate histidine ligation. However, further MCD comparisons of chlorin-reconstituted myoglobin with parallel ligand-bound adducts of the catalase clearly rule out histidine ligation in the latter, leaving tyrosinate as the best candidate for the proximal ligand.

Binding Sites↗

PubMed1991

Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII.

A 3,466-bp nucleotide sequence containing the katE gene of Escherichia coli has been determined. An open reading frame of 2,259 bp was found and was preceded by a potential ribosome-binding site. The predicted N-terminal sequence agreed with the sequence determined by direct amino acid sequencing, and the predicted direction of transcription was confirmed by expression of the gene cloned in both directions behind a T7 promoter. The start site of transcription was determined to be 127 bp upstream from the start of the open reading frame, and a potential RNA polymerase-binding site similar to a sequence preceding the xthA gene, which is also controlled by the KatF protein, was identified. The predicted sequence of the 753-amino-acid protein was compared with known sequences of other catalases, revealing significant similarity to the shorter catalases, including the residues in the putative active site and residues involved in heme binding.

Amino Acid Sequence↗

PubMed1990

Crystallization and preliminary X-ray diffraction analysis of catalase HPII from Escherichia coli.

Green crystals of the hexameric catalase HPII from Escherichia coli have been obtained by the hanging-drop method. The crystals belong to the monoclinic space group P2 with a = 123 A, b = 132 A, c = 93 A, beta = 112.5 degrees. There are three subunits in the asymmetric unit. The crystals diffract at least to 3.2 A resolution and are suitable for further X-ray diffraction studies.

Catalase↗