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PubMed1996

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase-peroxidase activities and isoniazid resistance.

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase-peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG-defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase-peroxidase which reduce catalase activity also decrease peroxidase activity.

Antibodies, Bacterial↗
PubMed2003

Outer sphere mutagenesis of Lactobacillus plantarum manganese catalase disrupts the cluster core. Mechanistic implications.

X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure9, 725-738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a micro 1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an 'open' form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km for H2O2 ( approximately 1.4 m) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.

Bacterial Proteins↗
PubMed2000

The heme-independent manganese-peroxidase activity depends on the presence of the C-terminal domain within the Streptomyces reticuli catalase-peroxidase CpeB.

Streptomyces reticuli produces a heme-containing homodimeric enzyme (160 kDa), the catalase-peroxidase CpeB, which is processed to the enzyme CpeC during prolonged growth. CpeC contains four subunits of 60 kDa each that do not include the C-terminal portion of the progenitor subunits. A genetically engineered cpeB gene encodes a truncated subunit lacking 195 of the C-terminal amino acids; four of these subunits assemble to form the enzyme CpeD. Heme binds most strongly in CpeB, least in CpeD. The catalase-peroxidase CpeB and its apo-form (obtained after extraction of heme) catalyze the peroxidation of Mn(II) to Mn(III), independent of the presence or absence of the heme inhibitor KCN. CpeC and CpeD, in contrast, do not exhibit manganese-peroxidase activity. The data show for the first time that a bacterial catalase-peroxidase has a heme-independent manganese-peroxidase activity, which depends on the presence of the C-terminal domain.

Amino Acid Sequence↗
PubMed2001

Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect.

Prolonged incubation of human spermatozoa can have deleterious effects on sperm function. The aim of this paper was to describe the effects of a prolonged in vitro incubation, under similar conditions to those employed in human assisted reproduction, on various sperm functional parameters, and to investigate the effect of an antioxidant (catalase) on this system. Freshly collected ejaculates from 20 healthy donors were studied. Samples were divided into two aliquots: the first was incubated with Ham's F10 containing 3.5% HAS, and the second was incubated in the same medium plus catalase (100 units ml-1). All experiments were carried out with spermatozoa isolated using the swim-up technique. Spermatozoa recovered from the supernatant after 1 h (T1) of incubation in 5% CO2 in air at 37 degrees C, and after 5 h (T6), 23 h (T24) and 47 h (T48), were evaluated for concentration, motion parameters including hyperactivation (computer-assisted analysis), viability, ATP concentration, reactive oxygen species (ROS) generation, DNA integrity (acridine orange), and acrosome reaction (AR). The major alteration observed in sperm function during the prolonged in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. ROS levels increased with the incubation time. No substantial modifications of sperm viability, chromatin condensation and AR inducibility were observed. The addition of catalase to the medium, while keeping ROS values within baseline levels, did not prevent the loss of motility or the corresponding increase in ATP.

Acrosome Reaction↗
PubMed1999

Synthesis of glucose oxidase and catalase by Aspergillus niger in resting cell culture system.

The synthesis of glucose oxidase and catalase by Aspergillus niger was investigated using a resting cell culture system without growth being established. Calcium carbonate induced the synthesis of both enzymes and calcium chloride inhibited it. The optimal pH for the biosynthesis of glucose oxidase and catalase was 6.0 and 5.7, respectively. The effects of other bivalent cations, reductive compounds and metabolic products on enzyme synthesis were also tested. The biosynthesis of glucose oxidase and catalase was promoted by MnCO3, thioglycolic acid, pyroracemic acid and gluconic acid.

Aspergillus niger↗
PubMed2002

Improvement in the resting-cell bioconversion of penicillin G to deacetoxycephalosporin G by addition of catalase.

AIMS: To improve the resting cell bioconversion of penicillin G to deacetoxycephalosporin G (DAOG) by elimination of an oxidizing intermediate which inactivates the enzyme during the reaction. METHODS AND RESULTS: Resting cells of Streptomyces clavuligerus strain NP1 were incubated with penicillin G, required co-factors and decane in the presence of catalase or superoxide dismutase, and production of DAOG was measured. Catalase stimulated the bioconversion but superoxide dismutase did not. CONCLUSIONS: Production of hydrogen peroxide during the ring expansion reaction is at least partially responsible for enzyme inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Catalase addition improves the bioconversion and will contribute to the eventual replacement of the current multi-step, expensive and environmentally-unfriendly chemical ring expansion by a biological route.

Alkanes↗
PubMed1995

Effect of peroxisomicine and related anthracenones on catalase activity.

Dimeric anthracenones were isolated from toxic plants of the genus Karwinskia (Rhamnaceae). T 514 or peroxisomicine A1 is one of these toxic compounds which produces an irreversible and selective damage on the peroxisomes of yeast cells in vivo. In this paper we now report the inhibitory effect in vitro of peroxisomicine A1 and other structurally related anthracenones on liver catalase activity. The peroxisomicine A1 produces a non-competitive inhibition with respect to H2O2 on bovine, dog, and mouse liver catalases. In the three cases Vmax was decreased whereas Km was unaffected. Other dimeric anthracenones of natural origin were also found to be inhibitors of bovine liver catalase. There is a relationship between structure and degree of inhibition of all anthracenonic compounds tested. Peroxisomicine A1 and peroxisomicine A2 caused the highest degree of inhibition (IC50 = 3.34 and 3.64 microM, respectively).

Animals↗
PubMed2004

The structure of coral allene oxide synthase reveals a catalase adapted for metabolism of a fatty acid hydroperoxide.

8R-Lipoxygenase and allene oxide synthase (AOS) are parts of a naturally occurring fusion protein from the coral Plexaura homomalla. AOS catalyses the production of an unstable epoxide (an allene oxide) from the fatty acid hydroperoxide generated by the lipoxygenase activity. Here, we report the structure of the AOS domain and its striking structural homology to catalase. Whereas nominal sequence identity between the enzymes had been previously described, the extent of structural homology observed was not anticipated, given that this enzyme activity had been exclusively associated with the P450 superfamily, and conservation of a catalase fold without catalase activity is unprecedented. Whereas the heme environment is largely conserved, the AOS heme is planar and the distal histidine is flanked by two hydrogen-bonding residues. These critical differences likely facilitate the switch from a catalatic activity to that of a fatty acid hydroperoxidase.

Animals↗
PubMed1990

Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity.

Melanogenesis is regulated in large part by tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmented (Blt/Blt) coat versus the wild-type black (B/B). We show that the b locus codes for a glycoprotein with the activity of a catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, we conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. Our studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the Blt mutation renders the protein susceptible to rapid proteolytic degradation.

Albinism↗
PubMed1990

Expression of the maize Cat3 catalase gene is under the influence of a circadian rhythm.

The accumulation of Cat3 catalase mRNA in the green leaves of maize (Zea mays) exhibits an unusual circadian rhythm. The steady-state level of the Cat3 transcript varies dramatically over the day in seedlings grown on a 12-hr photoperiod. Low or undetectable levels of the Cat3 mRNA are found late in the dark and early in the light period, while the transcript accumulates to high levels late in the light and early in the dark period. This dramatic diurnal variation in mRNA level does not occur with the maize Cat1 or Cat2 catalase transcripts. The diurnal fluctuation in Cat3 mRNA persists when the seedlings are transferred to continuous light or darkness, which indicates the influence of a circadian rhythm. The lack of influence of red and/or far-red light on this diurnal variation in transcript level indicates that the circadian rhythm is not regulated by phytochrome. Run-on transcription assays show that changes in the rate of Cat3 gene transcription closely parallel the observed changes in steady-state mRNA levels. This circadian rhythm in Cat3 gene expression and the resulting high levels of mRNA which accumulate late in the light period suggest that the catalase 3 (CAT-3) isozyme is being synthesized for accumulation in the dark period. This, together with the high level of Cat3 mRNA and CAT-3 protein accumulation in dark-grown maize shoots, suggests that the activity of the CAT-3 isozyme might be associated with a metabolic process important in shoot cells in the dark.

Catalase↗
PubMed1995

Spectral characterization and chemical modification of catalase-peroxidase from Streptomyces sp.

Catalase-peroxidase was purified to near homogeneity from Streptomyces sp. The enzyme was composed of two subunits with a molecular mass of 78 kDa and contained 1.05 mol of protoporphyrin IX/mol of dimeric protein. The absorption and resonance Raman spectra of the native and its cyano-enzyme were closely similar to those of other heme proteins with a histidine as the fifth ligand. However, the peak from tyrosine ring at approximately 1612 cm-1, which is unique in catalases, was not found in resonance Raman spectra of catalase-peroxidase. The electron paramagnetic resonance spectrum of the native enzyme revealed uniquely two sets of rhombic signals, which were converted to a single high spin, hexacoordinate species after the addition of sodium formate. Cyanide bound to the sixth coordination position of the heme iron, thereby converting the enzyme to a low spin, hexacoordinate species. The time-dependent inactivation of the enzyme with diethyl pyrocarbonate and its kinetic analysis strongly suggested the occurrence of histidine residue. From the above-mentioned spectroscopic results and chemical modification, it was deduced that the native enzyme is predominantly in the high spin, ferric form and has a histidine as the fifth ligand.

Catalase↗
PubMed1997

Thermal conversion from low- to high-activity forms of catalase I from Bacillus stearothermophilus.

Catalase I from Bacillus stearothermophilus has the interesting property of increasing its enzyme activity on heating. It was confirmed that after heating at 70 degrees C for 10 min or 65 degrees C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form. The increase in kcat from 1400 to 3930 s-1 and the decrease in Km for H2O2 from 4.4 to 2.7 mM by heat activation indicate changes in the kinetic property of the enzyme molecule. Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form. The heat activation process followed the first-order kinetics with an activation enthalpy (DeltaH*) of 191 kJ/mol while the heat denaturation process had a DeltaH* of 545 kJ/mol. The CD spectra of the two enzyme forms had small but marked differences. The conversion of the low-activity form to the high-activity form was an endothermic process with a Tm of 56 degrees C, which is much lower than that of the heat denaturation (Tm = 76 degrees C), and the enthalpy change for the transition was only 5% of that for the denaturation. It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme. In addition, the newly obtained low-activity form was brought to a high-activity form by heating. These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier.

Calorimetry, Differential Scanning↗
PubMed1997

Overexpression, purification, and characterization of the catalase-peroxidase KatG from Mycobacterium tuberculosis.

Wild-type catalase-peroxidase KatG from Mycobacterium tuberculosis as well as a specific mutant (R463L) frequently found in isoniazid-resistant strains have been overexpressed in Escherichia coli, allowing purification of sufficient quantities of enzyme for physical and kinetic characterization. Optical absorption and EPR spectroscopies indicate that KatG is similar to a growing class of bacterial catalase-peroxidases. Optical and EPR spectra of KatG in the presence of either a strong field or weak field ligand suggest that, like horseradish peroxidase and metmyoglobin, KatG is likely to have a histidine as a proximal ligand. The wild-type enzyme functions as a highly active catalase as well as a broad specificity peroxidase. Wild-type KatG and the R463L mutant of KatG exhibit identical spectroscopic and kinetic properties. Furthermore, both enzymes are equally capable of metabolizing the important antituberculosis drug isoniazid.

Bacterial Proteins↗
PubMed2002

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation.

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.

Catalase↗
PubMed2005

The Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG): autocatalytic formation and effect on enzyme catalysis and spectroscopic properties.

Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met(255)-Tyr(229)-Trp(107) cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P.)Fe(III), where P. represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.

Amino Acid Sequence↗
PubMed2005

Role of the main access channel of catalase-peroxidase in catalysis.

Catalase-peroxidases (KatG) are bifunctional heme peroxidases with an overwhelming catalatic activity. The structures show that the buried heme b is connected to the exterior of the enzyme by a main channel built up by KatG-specific loops named large loop LL1 and LL2, the former containing the highly conserved sequence Met-Gly-Leu-Ile-Tyr-Val-Asn-Pro-Glu-Gly. LL1 residues Ile248, Asn251, Pro252, and Glu253 of KatG from Synechocystis are the focus of this study because of their exposure to the solute matrix of the access channel. In particular, the I248F, N251L, P252A, E253Q, and E253D mutants have been analyzed by UV-visible and resonance Raman spectroscopies in combination with steady-state and presteady-state kinetic analyses. Exchange of these residues did not alter the kinetics of cyanide binding or the overall peroxidase activity. Moreover, the kinetics of compound I formation and reduction by one-electron donors was similar in the variants and the wild-type enzyme. However, the turnover numbers of the catalase activity of I248F, N251L, E253Q, and E253D were only 12.3, 32.6, 25, and 42% of the wild-type activity, respectively. These findings demonstrate that the oxidation reaction of hydrogen peroxide (not its reduction) was affected by these mutations. The altered kinetics allowed us to monitor the spectral features of the dominating redox intermediate of E253Q in the catalase cycle. Resonance Raman data and structural analysis demonstrated the existence of a very rigid and ordered structure built up by the interactions of these residues with distal side and also (via LL1) proximal side amino acids, with the heme itself, and with the solute matrix in the channel. The role of Glu253 and the other investigated channel residues in maintaining an ordered matrix of oriented water dipoles, which guides hydrogen peroxide to its site of oxidation, is discussed.

Amino Acid Sequence↗
PubMed2001

Enzymatic and non-enzymatic reduction of brucine N-oxide by aldehyde oxidase and catalase.

1. Brucine N-oxide was reduced by aldehyde oxidase in rabbit liver cytosol in the presence of an electron donor, 2-hydroxypyrimidine, under anaerobic conditions. The flavoprotein purified from rabbit liver exhibited significant reductase activity in the presence of electron donors. 2. Brucine N-oxide was also reduced by rabbit liver cytosol and blood in the presence of both a reduced pyridine nucleotide and FAD under anaerobic conditions. The N-oxide reductase activities were inhibited by carbon monoxide and air. However, these activities were not abolished whe n liver cytosol and blood were boiled. Rabbit erythrocytes exhibited the reductase activity, but not plasma. 3. When liver cytosol or blood was separated by DEAE-cellulose column chromatography, the fractions with the reducing activity in the presence of both NADH and FAD also showed catalase activity. 4. Catalase catalysed the brucine N-oxide reduction in the presence of both NAD(P)H and FAD. Hematin also exhibited the reductase activity in the presence of both NAD(P)H and FAD. Photochemically reduced FAD was effective in the reduction instead of NAD(P)H and FAD. 5. Bricine N-oxide reduction proceeds via two routes in liver cytosol and blood. One is enzymatic reduction by aldehyde oxidase; the other is non-enzymatic reduction catalysed by the haem group of catalase in the presence of reduced flavin.

Aldehyde Oxidase↗
PubMed1983

The role of cellular catalase on the radiosensitization of bacterial vegetative cells by N2O.

The effect of N2O on eight strains of bacteria was measured in dilute suspensions. The dose-modifying factors (DMF) of N2O on M. radiodurans R1, P. radiora 0-1, M. lysodeikticus and B. pumilus E601 (vegetative cells) were 3 . 4, 2 . 9, 2 . 4 and 1 . 7, respectively. But P. radiora RP-C, P. fluorescens B3-1, E. coli B/r and E. coli K-12 were hardly sensitized by N2O. From measurements of catalase activity of each bacterium, it was found that the DMF increases with increased catalase activity, suggesting that cellular catalase promotes the sensitizing action of N2O.

Bacteria↗