Biomedical subjects

J Switala

Publications and source records attributed to J Switala.

At least 19 recordsLinked to original sources

PubMed2001

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.

Alleles↗

PubMed2001

Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeri.

Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P2(1), with unit-cell parameters a = 60.6, b = 153.9, c = 109.2 A, beta = 102.8 degrees. From these crystals a diffraction data set to 1.8 A resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5 A. These crystals diffracted beyond 2.2 A resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis.

Catalase↗

PubMed2001

The role of distal tryptophan in the bifunctional activity of catalase-peroxidases.

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

Bacterial Proteins↗

PubMed1999

Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli.

The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered.

Amino Acid Sequence↗

PubMed1999

Catalase HPII from Escherichia coli exhibits enhanced resistance to denaturation.

Catalase HPII from Escherichia coli is a homotetramer of 753 residue subunits. The multimer displays a number of unusual structural features, including interwoven subunits and a covalent bond between Tyr415 and His392, that would contribute to its rigidity and stability. As the temperature of a solution of HPII in 50 mM potassium phosphate buffer (pH 7) is raised from 50 to 92 degrees C, the enzyme begins to lose activity at 78 degrees C and 50% inactivation has occurred at 83 degrees C. The inactivation is accompanied by absorbance changes at 280 and 407 nm and by changes in the CD spectrum consistent with small changes in secondary structure. The subunits in the dimer structure remain associated at 95 degrees C and show a significant level of dissociation only at 100 degrees C. The exceptional stability of the dimer association is consistent with the interwoven nature of the subunits and provides an explanation for the resistance to inactivation of the enzyme. For comparison, catalase-peroxidase HPI of E. coli and bovine liver catalase are 50% inactivated at 53 and 56 degrees C, respectively. In 5.6 M urea, HPII exhibits a coincidence of inactivation, CD spectral change, and dissociation of the dimer structure with a midpoint of 65 degrees C. The inactive mutant variants of HPII which fold poorly during synthesis and which lack the Tyr-His covalent bond undergo spectral changes in the 78 to 84 degrees C range, revealing that the extra covalent linkage is not important in the enhanced resistance to denaturation and that problems in the folding pathway do not affect the ultimate stability of the folded structure.

Catalase↗

PubMed1999

Structure of catalase HPII from Escherichia coli at 1.9 A resolution.

Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase. The structure of native HPII has been refined at 1.9 A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are respectively 16.6% and 21.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 A for 477 equivalencies with beef liver catalase. Most of the additional 276 residues of HPII are located in either an extended N-terminal arm or in a C-terminal domain organized with a flavodoxin-like topology. A small number of mostly hydrophilic interactions stabilize the relative orientation between the C-terminal domain and the core of the enzyme. The heme component of HPII is a cis-hydroxychlorin gamma-spirolactone in an orientation that is flipped 180 degrees with respect to the orientation of the heme found in beef liver catalase. The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cbeta atom and the Ndelta atom of His392. Over 2,700 well-defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule. Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions. The longest channel, that begins in an adjacent subunit, is over 50 A in length, and the second channel is about 30 A in length. A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His-Tyr bond formation. HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585-590, corresponds to the entrance to the second channel. The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination.

Amino Acid Sequence↗

PubMed1999

Role of the lateral channel in catalase HPII of Escherichia coli.

The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.

Alanine↗

PubMed1998

Truncation and heme pocket mutations reduce production of functional catalase HPII in Escherichia coli.

The subunit of catalase HPII from Escherichia coli is 753 residues in length and contains a core of approximately 500 residues, with high structural similarity to all other heme catalases. To this core are added extensions of approximately 80 and 180 residues at the N- and C-termini, respectively. The tetrameric structure is made up of a pair of interwoven dimers in which 90 N-terminal residues of each subunit are inserted through a loop formed by the hinge region linking the beta-barrel and alpha-helical domains of the adjacent subunit. A high concentration of proline residues is found in the vicinity of the overlap regions. To study the influence of the extended regions on folding and subunit association of HPII, a diversity of modifications have been introduced. Removal of the complete C-terminal domain or the N-terminal extension, either separately or together, effectively creating a small subunit catalase, resulted in no enzyme accumulation. Systematic truncations showed that only nine C-terminal residues (Ile745 to Ala753) could be removed without significantly affecting the accumulation of active enzyme. Removal or even conservative replacements of the side chain of Arg744 significantly reduced the accumulation of active enzyme despite this residue interacting only with the C-terminal domain. Removal of as few as 18 residues from the N-terminus also reduced accumulation of active enzyme. Changes to other residues in the protein, including residues in the heme binding pocket, also reduced the accumulation of active protein without substantially affecting the enzyme specific activity. Implications of these data for the interdependence of subunit folding and subunit-subunit interactions are discussed.

Catalase↗

PubMed1998

Isometric muscle strength in alcoholic and nonalcoholic liver-transplantation candidates.

Intensity of isometric muscle contractions was measured in alcoholic subjects with cirrhosis (N = 42), nonalcoholic subjects with cirrhosis (N = 33), and normal controls (N = 31). Muscle strength and endurance were comparable in the alcoholic and nonalcoholic cirrhotic subjects for all variables. Both cirrhotic groups were inferior to normal controls for all variables. The quantity x frequency (Q x F) index reported for the period during peak alcohol consumption correlated with 6 muscle-force variables, accounting for 9-20% of the variance. Alcoholic and nonalcoholic cirrhotic subjects did not differ in Quality of Life Inventory (QOLI) scales. Significant correlations, however, were found for the alcoholic cirrhotic subjects but not for the nonalcoholic cirrhotic subjects between quality-of-life indices and muscle strength and endurance. Muscle weakness is thus differentially associated with quality of life in alcoholic cirrhotic subjects as compared with nonalcoholic subjects with cirrhosis even though level of strength and endurance in the two groups is comparable.

Adult↗

PubMed1997

Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli.

A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed.

Amino Acid Sequence↗

PubMed1997

Isokinetic muscle strength and its association with neuropsychological capacity in cirrhotic alcoholics.

Alcoholic cirrhotics (n = 49), nonalcoholic cirrhotics (n = 42), and normal controls (n = 50) were compared on measures of isokinetic muscle strength and neuropsychological capacity. Alcoholic cirrhotics were deficient on measures of eccentric and concentric muscle movements, compared with normal controls but were not different from nonalcoholic cirrhotics. Nor were differences observed between the two cirrhotic groups on neuropsychological tests of cognitive and psychomotor capacity, suggesting that cirrhosis rather than alcoholism per se is responsible for the manifest deficits. Psychomotor capacity correlated negatively with isokinetic strength in cirrhotic subjects. These findings suggest that muscle weakness, due either directly to advanced liver disease or mediated by subclinical hepatic encephalopathy, accounts for a portion of the variance on the neuropsychological test performance of cirrhotic alcoholics.

Adult↗

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

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

Early interactions between drug-involved mothers and infants. Within-group differences.

OBJECTIVE: To explore differences in maternal characteristics, mother-infant interaction, and infant development within a group of women who used cocaine, alcohol, and tobacco during pregnancy and their infants. DESIGN: Prospective survey. SETTING: Countywide, voluntary, home-based clinical intervention program. PARTICIPANTS: Thirty-two mother-infant pairs identified through a risk-assessment screen who participated in the program for 1 year. VARIABLES: Maternal characteristics, neonatal characteristics, interactional measures (Nursing Child Assessment Feeding Scale and Home Observation for Measurement of the Environment scale), and developmental assessment (Bayley Scales of Infant Development at 1 year). RESULTS: The majority of women were black, single, and unemployed. Fifteen (47%) of the infants were born prematurely; four (13%) were small for gestational age. Mean Bayley Scales of Infant Development scores were as follows: the mental development index was 99.8, and the psychomotor development index was 102.4. Older mothers (r = .41, P = .04), mothers of higher parity (r = .42, P = .02), and mothers who were more actively involved in the program (r = .41, P = .04) had higher scores on the Nursing Child Assessment Satellite Training Feeding Scale. Mothers who were better educated (r = .49, P = .009) and mothers who were more active in the program (r = .44, P = .02) had higher scores on the Home Observation for Measurement of the Environment scale. Several of the subscales of the Home Observation for Measurement of the Environment scale were significantly associated with scores on the Bayley Scales of Infant Development. CONCLUSIONS: For this group of substance-exposed infants whose mothers were receiving support services, developmental skills at 1 year were age appropriate. Despite drug abuse and poverty, there was some variability in the ability of mothers to provide a developmentally supportive environment for their infants. Those who were better organized to support infant development had infants who performed better on global developmental assessments.

Child Development↗

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↗

PubMed1995

Abstracting capacity in cirrhotic alcoholics: negative findings.

OBJECTIVE: Alcoholic and nonalcoholic cirrhotics and normal controls were compared to determine the extent to which the cognitive deficits frequently observed in alcoholics are attributable to hepatic encephalopathy. METHOD: A battery of neuropsychological tests was administered measuring verbal and nonverbal abstracting capacity. RESULTS: No significant differences between the three groups were observed. Both the alcoholic (n = 43) and nonalcoholic (n = 63) subjects performed comparably to normal controls (n = 21) and the former two groups performed comparably to each other on seven indicators of abstracting capacity. CONCLUSIONS: Deficits in reasoning ability are not invariably associated with chronic alcoholism. In addition, low grade hepatic encephalopathy concomitant to cirrhosis in both alcoholics and nonalcoholics does not impact adversely on abstracting capacity.

Adult↗

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↗