Isolation and expression of the catA gene encoding the major vegetative catalase in Streptomyces coelicolor Müller.

We isolated the catA gene for the major vegetative catalase from Streptomyces coelicolor Müller. It encodes a polypeptide of 488 residues (55,440 Da) that is highly homologous to typical monofunctional catalases. We investigated catA expression by analyzing both catA mRNA and catalase activity. catA expression was increased by H2O2 treatment but did not increase during stationary phase. A putative catalase (CatB) cross-reactive with anti-CatA antibody appeared during stationary phase and in the aerial mycelium.     

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.     

Purification and characterization of a catalase-peroxidase and a typical catalase from the bacterium Klebsiella pneumoniae

The bacterium Klebsiella pneumoniae synthesizes three different types of catalase: a catalase-peroxidase, a typical catalase and an atypical catalase, designated KpCP, KpT and KpA, respectively (Goldberg, I. and Hochman, A. (1989) Arch. Biochem. Biophys. 268, 124-128). KpCP, but not the other two enzymes, in addition to the catalatic activity, catalyzes peroxidatic activities with artificial electron donors, as well as with NADH and NADPH. Both KpCP and KpT are tetramers, with heme IX as a prosthetic group, and they show a typical high-spin absorption spectrum which is converted to low-spin when a cyanide complex is formed. The addition of dithionite to KpCP causes a shift in the absorption maxima typical of ferrous heme IX. KpCP has a pH optimum of 6.3 for the catalatic activity and 5.2-5.7 for the peroxidatic activity, and relatively low 'Km' values: 6.5 mM and 0.65 H2O2 for the catalatic and peroxidatic activities, respectively. The activity of the catalase-peroxidase is inhibited by azide and cyanide, but not by 3-amino-1,2,4-triazole. KpT has wide pH optimum: 5-10.5 and a 'Km' of 50 mM H2O2, it is inhibited by incubation with 3-amino-1,2,4-triazole and by the acidic forms of cyanide and azide. A significant distinction between the typical catalase and the catalase-peroxidase is the stability of their proteins: KpT is more stable than KpCP to H2O2, temperature, pH and urea.     

Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases

The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis. Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified. An A. fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe. Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli. Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified. These results indicate that A. fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB. The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A. fumigatus. Staining was observed heterogeneously throughout the fungal hyphae. This result indicates that catalase is produced by A. fumigatus during invasive aspergillosis.     

Cloning and Characterization of Catalases from Rice,Oryza sativa L.

Catalase is the major H₂O₂-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)₆-tagged proteins in Escherichia coli. The recombinant (His)₆-polypeptides were enriched to apparent homogeneity and characterized. With H₂O₂ as substrate, the highest catalase k _cat value (20±1.71×10^?3 min^?1) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.     

Competitiveness in root colonization by Pseudomonas putida requires the rpoS gene

The rpoS gene in Pseudomonas putida was essential for plant root colonization under competitive conditions from other microbes. The RpoS- mutant survived less well than the wild-type strain in culture medium, and unlike the wild-type, failed to colonize the roots in a peat matrix containing an established diverse microflora. The RpoS-deficient P. putida isolate was generated by insertion of a glucuronidase-npt cassette into the rpoS gene. The RpoS mutant had dose-dependent increased sensitivity to oxidative stress and produced Mn-superoxide dismutase activity earlier than the parent. While extracts from wild-type P. putida stationary-phase cells contained three isozymes of catalase (CatA, CatB, and CatC), the sigma38-deficient P. putida lacked CatB. These results are consistent with previous findings that CatB is induced in stationary-phase.     

Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease

Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.     

Purification and characterization of a novel type of catalase from the bacterium Klebsiella pneumoniae

A novel type of catalase, designated KpA, was purified from the bacterium Klebsiella pneumoniae. The enzyme is unique in that it is a dimer with subunit molecular weight of 80,000, it bears a chlorine-type heme as prosthetic group, and is active over a very wide range of H+ concentrations, with a plateau from pH 2.8 to 11.8. Yet, some properties of KpA are characteristic of typical catalases: it is stable when treated with with ethanol/chloroform, cannot be reduced by dithionite and it is inhibited by 3-amino-1,2,4-triazole and by the conjugate acid forms of azide and cyanide. The protein of KpA is outstandingly resistant to denaturing conditions: it retains full activity when incubated with 8 M urea, at 30 degrees C for 4 days, it is stable for 1 h at 70 degrees C and at pH values 3.1 and 11.5 and, when dialyzed against 50 mM H2O2, it still retains 42% of its activity after 80 min.     

免疫信号轴揭示水稻与病原菌斗争的秘密

近年,我国学者在水稻(Oryza sativa)广谱抗病机制、抗性与产量协调机制等方向的研究取得了一系列突破性进展。最近,何祖华和杨卫兵团队在水稻抗病性研究中再次取得突破,发现1个以钙感应蛋白ROD1和过氧化氢酶CatB为核心的信号轴,通过负调控水稻免疫反应,保证正常生长条件下维持低水平的免疫反应,促进水稻正常生长发育...     

Differential Accumulation of Salicylic Acid and Salicylic Acid-Sensitive Catalase in Different Rice Tissues

We previously proposed that salicylic acid (SA)-sensitive catalases serve as biological targets of SA in plant defense responses. To further examine the role of SA-sensitive catalases, we have analyzed the relationship between SA levels and SA sensitivity of catalases in different rice (Oryza sativa) tissues. We show here that, whereas rice shoots contain extremely high levels of free SA, as previously reported (I. Raskin, H. Skubatz, W. Tang, B.J.D. Meeuse [1990] Ann Bot 66: 369-373; P. Silverman, M. Seskar, D. Kanter, P. Schweizer, J.-P. Metraux, I. Raskin [1995] Plant Physiol 108: 633-639), rice roots and cell-suspension cultures have very low SA levels. Catalases from different rice tissues also exhibit differences in sensitivity to SA. Catalase from rice shoots is insensitive to SA, but roots and cell-suspension cultures contain SA-sensitive catalase. The difference in SA sensitivity of catalases from these different tissues correlates with the tissue-specific expression of two catalase genes, CatA and CatB, which encode highly distinctive catalase proteins. CatA, which encodes a catalase with relatively low sequence homology to the tobacco SA-sensitive catalases, is expressed at high levels exclusively in the shoots. On the other hand, in roots and cell-suspension cultures, with northern analysis we detected expression of only the CatB gene, which encodes a catalase with higher sequence homology to tobacco catalases. The role of catalases in mediating some of the SA-induced responses is discussed in light of these results and the recently defined mechanisms of catalase inhibition by SA.     

Purification,characterization, and gene sequencing of a catalase from an alkali- and halo-tolerant bacterium,Halomonas sp. SK1

An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4% yield of the catalase was obtained with 97% purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH2OH, NaN3, and 3-amino-1,2,4-triazole, active at pH 5.0-11.0, thermo-sensitive, and KCl-tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99% identity with that of Vibrio rumoiensis S-1T.     

Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein

Using chemical inhibitors and small interfering RNA (siRNA), we have confirmed roles for cathepsin B (CatB) and cathepsin L (CatL) in Ebola virus glycoprotein (GP)-mediated infection. Treatment of Ebola virus GP pseudovirions with CatB and CatL converts GP1 from a 130-kDa to a 19-kDa species. Virus with 19-kDa GP1 displays significantly enhanced infection and is largely resistant to the effects of the CatB inhibitor and siRNA, but it still requires a low-pH-dependent endosomal/lysosomal function. These and other results support a model in which CatB and CatL prime GP by generating a 19-kDa intermediate that can be acted upon by an as yet unidentified endosomal/lysosomal enzyme to trigger fusion.     

The Dictyostelium discoideum prespore-specific catalase B functions to control late development and to protect spore viability

Changes in the levels of reactive oxygen species (ROS) have been associated previously with cell differentiation and development in several systems. Thus, there is interest in studying the developmental regulation of antioxidant enzymes, whose activities may modulate ROS levels and subsequent oxidant-mediated signal transduction events in specific tissues. Our recent identification in Dictyostelium discoideum of the prespore-specific catalase B (CatB) enzyme suggested (a) that the CatB enzyme functions to provide protection to the mature spores, and (b) that the CatB enzyme may have a regulatory role in cell differentiation and morphogenesis. We have now confirmed both these hypotheses. We specifically disrupted the catB gene by homologous recombination. The resulting catB null strain displays a 4-h delay in development at the time of normal catB gene expression, followed by slow and asynchronous development of fruiting bodies, taking 10 h longer than the isogenic parent strain. The expression of both prestalk- and prespore-specific genes was altered in the mutant both temporally and quantitatively, and the resultant mutant spores had increased sensitivity to H(2)O(2). This study supports the idea that CatB functions in the development of D. discoideum by regulating the level of ROS, and adds to the growing body of evidence for regulatory roles for ROS.     

Multiple catalase genes are differentially regulated in Aspergillus nidulans

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes, catA and catB, have been studied in Aspergillus nidulans. Here we have characterized a third catalase gene, designated catC, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi, Archaea, and animals. In contrast, the CatA (approximately 84 kDa) and CatB (approximately 79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The catC gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of catC eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in catA catB mutant strains during late stationary phase was still present in catC and catABC null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither catC nor catABC triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H(2)O(2)-generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H(2)O(2) treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H(2)O(2) detoxification in filamentous fungi.     

Cathepsin B Degrades Amyloid-β in Mice Expressing Wild-type Human Amyloid Precursor Protein

Accumulation of amyloid-β (Aβ), believed to be a key trigger of Alzheimer disease (AD), could result from impaired clearance mechanisms. Previously, we showed that the cysteine protease cathepsin B (CatB) degrades Aβ, most likely by C-terminal truncation, in mice expressing human amyloid precursor protein with familial AD-linked mutations (hAPP_FAD). In addition, the Aβ-degrading activity of CatB is inhibited by its endogenous inhibitor, cystatin C (CysC). Reducing CysC expression markedly lowers Aβ levels by enhancing CatB-mediated Aβ degradation in hAPP_FAD mice. However, because a vast majority of AD patients do not carry familial mutations, we investigated how the CysC-CatB axis affects Aβ levels in mice expressing wild-type hAPP (hAPP_WT). Enhancing CatB activity by CysC deletion significantly lowered total Aβ and Aβ42 levels in hAPP_WT mice, whereas CatB deletion increased Aβ levels. To determine whether neuron-derived CatB degrades Aβ in vivo, we generated transgenic mice overexpressing CatB under the control of a neuron-specific enolase promoter. Enhancing neuronal CatB activity in hAPP_WT mice significantly lowered Aβ42 levels. The processing of hAPP_WT was unaffected by increasing or ablating CatB activity. Thus, the CysC-CatB axis affects degradation of Aβ42 derived from hAPP lacking familial mutations. These findings support the notion that enhancing CatB activity could lower Aβ, especially Aβ42, in AD patients with or without familial mutations.     

Purification and characterization of a rat liver ferroactivator with catalase activity

A rat liver protein with both phosphoenolpyruvate carboxykinase ferroactivator activity and catalase activity has been purified to near-homogeneity. The protein has a native molecular weight of 240,000 and is composed of four identical subunits containing ferriprotoporphyrin IX prosthetic groups. The visible spectrum has absorbance maxima at 403, 500, 530, and 620 nm; it is not reduced by dithionite. The spectrum, physical properties, and specific activity are almost identical with those of catalases from other sources, and the protein has been tentatively identified as rat liver catalase. The protein exhibited partial reactivity in double immunodiffusion plates to antiserum prepared against rat liver ferroactivator isolated by a previous method (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440) raising the possibility that the original ferroactivator and rat liver catalase are structurally related. Inactivation of catalase by 3-amino-1,2,4-triazole was accompanied by loss of ferroactivator activity as well. The apparent specific activity of ferroactivator, as well. The apparent specific activity of ferroactivator, whether heme-containing or not, can be increased between 2- and 100-fold by the inclusion of bovine serum albumin, HCO3-, or a combination of the two in the incubation.     

Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.     

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.     

Menadione-resistant Chinese hamster cell variants are cross-resistant to hydrogen peroxide and exhibit stable chromosomal and biochemical alterations

We have investigated the antioxidant properties of V79 Chinese hamster cells rendered resistant to menadione by chronic exposure to increasing concentrations of this quinone. MD1, a clone of resistant cells, was compared to the parental M8 cells; the former showed increased activity of catalase (3 fold), glutathione peroxidase (1.6 fold) and DT-diaphorase (2.6 fold), as well as an increase in glutathione (3.2 fold). Although one of the products of menadione metabolism is superoxide anion, no changes in total superoxide dismutase activity was observed in MD1 cells. MD1 menadione resistant cells were also resistant to killing by hydrogen peroxide and contained tandem duplication of chromosome 6. A similar duplication of chromosome 6 was seen in several independently derived menadione resistant clones and therefore seems closed linked to the establishment of the resistance. Upon removal of menadione from the medium, some of these properties of MD1 cells, viz., resistance to menadione, elevated glutathione levels, and glutathione peroxidase activity, were lost and the cells resembled M8 cells. However, resistance to H₂O₂, elevated catalase activity and the duplicated chromosome remained stable for more than 40 cell passages in the absence of menadione. The increase in catalase activity was correlated with an increase in catalase mRNA content and a 50% amplification of catalase gene, as determined, respectively, by Northern and Southern blot analysis. The role of the chromosome 6 duplication in resistance to oxidative stress remains to be established. It is not responsible directly for elevated catalase levels since the catalase gene is on chromosome 3.Abbreviations SDS Sodium Dodecyl Sulphate - SOD Superoxide Dismutase - PBS Phosphate Buffered Saline (8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 137 mM NaCl) - CDTA N,N,N,N-tetracetic-trans-1,2-diaminocyclohexane acid - MOPS Sulphonic-3-(N-morpholine)-propane acid - SSC 150 mM Nacl, 15 mM sodium-citrate, pH 6.8