H2O2 and cGMP may function as an O2 sensor in the pulmonary artery

The effects of O2 tension on force in precontracted isolated pulmonary arterial smooth muscle from calf lungs was characterized to investigate the mechanism of O2 tension sensing. These arteries display a decrease in force with increasing O2 tension that is antagonized via inhibition of soluble guanylate cyclase activation by 10 microM methylene blue or inactivation of catalase by pretreatment with 50 mM 3-amino-1,2,4-triazole for 30 min. O2 tension-dependent relaxation is associated with an increase in intracellular H2O2 metabolism through catalase (detected as the peroxide-dependent inactivation of tissue catalase activity by aminotriazole) and cyclic guanosine 5'-monophosphate (cGMP), known mediators of relaxation in calf pulmonary arteries. Thus a recently reconstructed mechanism of activation of soluble guanylate cyclase involving the metabolism of H2O2 by catalase appears to function as an O2 tension sensor in pulmonary arteries.     

Fatty acid-dependent ethanol metabolism

Rates of ethanol oxidation by perfused livers from fasted female rats were decreased from 82 +/- 8 to 11 +/- 7 mumol/g/hr by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. The subsequent addition of fatty acids of various chain lengths in the presence of 4-methylpyrazole increased rates of ethanol uptake markedly. Palmitate (1 mM) increased rates of ethanol oxidation to 95 +/- 8 mumol/g/hr, while octanoate and oleate increased rates to 58 +/- 11 and 68 +/- 15 mumol/g/hr, respectively. Hexanoate, a short-chain fatty acid oxidized predominantly in the mitochondria, had no effect. Addition of oleate also increased the steady-state level of catalase-H2O2. Pretreatment of rats for 1.5 hours with 3-amino-1,2,4-triazole (1.0 g/kg), an inhibitor of catalase, prevented the ethanol-dependent decrease in the steady-state level of catalase-H2O2 completely. Under these conditions, aminotriazole decreased rates of ethanol oxidation by about 50% and blocked the stimulation of ethanol oxidation by fatty acids. Oleate decreased rates of aniline hydroxylation by about 50%, indicating that cytochrome P450 is not involved in the stimulation of ethanol uptake by fatty acids. Furthermore, oleate stimulated ethanol uptake in livers from ADH-negative deermice indicating that fatty acids do not simply displace 4-methylpyrazole from alcohol dehydrogenase. It is concluded that the stimulation of ethanol oxidation by fatty acids is due to increased H2O2 supplied by the peroxisomal beta-oxidation of fatty acids for the catalase-H2O2 peroxidation pathway.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Inhibition of catalase by 3,3''-diaminobenzidine.

3,3'-Diaminobenzidine strongly inhibits bovine liver catalase in two distinct ways. One of these was competitive with respect to H2O2, approached a limit of 100% inhibition and was rapidly reversed by dilution or by dialysis. The other was dependent upon H2O2, approached a limit of 60% and was not reversed by dilution or dialysis. Exposure to diaminobenzidine followed by dialysis did not modify the electrophoretic mobility of the enzyme, its Km for H2O2 or its optical spectrum, although Vmax. was halved. Diaminobenzidine protects catalase against the irreversible inactivation imposed by 3-amino-1,2,4-triazole plus H2O2. CN-protected catalase against the apparently irreversible inhibition by diaminobenzidine, and dialysis against CN- reversed this inhibition. Ethanol was similarly protective, and ethanol plus H2O2 reversed the inhibition by diaminobenzidine. Several other aromatic diamines inhibited in a fashion similar to that of diaminobenzidine, but were less effective. A scheme of reactions that accounts for these observations is proposed.     

Peroxisomal beta-oxidation from endogenous substrates. Demonstration through H2O2 production in the unanaesthetized mouse.

A system was developed in which it is possible to detect in vivo changes in hepatic H2O2 production, using a combination of the catalase inhibitor, 3-amino-1,2,4-triazole and methanol. In mice, starvation significantly increases hepatic H2O2 production and plasma non-esterified fatty acid concentrations. Short-term refeeding after a 24 h starvation period brings H2O2 production and plasma non-esterified fatty acid concentration back to normal in 3h. Administration of insulin 24 h after the onset of starvation normalizes H2O2 production in less than 2h and decreases non-esterified fatty acid concentration below normal values. The suppression by insulin of H2O2 production, as well as its coherence with plasma non-esterified fatty acid concentration, indicate that increased H2O2 production in starved mice reflects peroxisomal beta-oxidation.     

Catalase-dependent measurement of H2O2 in intact mitochondria

Mitochondria generate potentially damaging amounts of superoxide and H2O2 during oxidative metabolism. Although many assays are available to measure mitochondrial H2O2 generation, most detect H2O2 that has escaped the organelle. To measure H2O2 within mitochondria that contain catalase, we have developed an assay based on the ability of H2O2 to inhibit catalase in the presence of 3-amino-1,2,4-triazole. The assay is simple to perform, does not require expensive instrumentation, and is specific for H2O2. Results from this assay show that H2O2 generation in rat heart mitochondria reflects the activity of the electron transport chain. Further, liver mitochondria prepared from selenium-deficient rats have increased succinate-stimulated rates of H2O2 generation. This indicates that mitochondrial selenoenzymes are important for H2O2 removal. It also demonstrates the utility of this assay in measuring H2O2 release from mitochondria that do not contain catalase. The assay should be useful for study of both superoxide-dependent H2O2 generation in situ, and the role of endogenous mitochondrial catalase in H2O2 removal.     

Superoxide, hydrogen peroxide, and oxygen toxicity in two free-living nematode species

Two species of free-living nematodes, Turbatrix aceti and Caenorhabditis elegans, exhibited a marked sensitivity to 3 atm of 100% O2. Environmental changes in pH and temperature, which altered nematode respiration, resulted in alterations in the survival of these organisms under high pO2. Levels of defensive enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and dianisidine peroxidase were measured in the two species. No changes in the level of superoxide dismutase or catalase activity were induced by exposure of the nematodes to high pO2. Manipulation of these two enzymes was however achieved using the inhibitors 3-amino-1,2,4-triazole and diethyldithiocarbamate. 3-Amino-1,2,4-triazole (20 mM) eliminated greater than or equal to 80% of the catalase activity in vivo and diethyldithiocarbamate (5 mM) decreased the level of CuZn superoxide dismutase by greater than or equal to 70%. Both of these compounds increased the sensitivity of C. elegans to high pO2 toxicity. Compounds capable of intracellular redox-cycling with O2- -production, such as plumbagin, increased CN- -resistant respiration in the nematodes and imposed an O2-dependent toxicity. These experiments demonstrate the toxicity of intracellular O2- and H2O2 in nematodes and the importance of superoxide dismutase and catalase in providing a defense against these toxic molecules in vivo.     

Catalase-Aminotriazole Method for Measuring Secretion of Hydrogen Peroxide by Microorganisms

A new method for measuring the secretion of H(2)O(2) has been based upon an H(2)O(2)-dependent inhibition of catalase by 3-amino-1,2,4-triazole. The conversion of an H(2)O(2)-secretion rate into a catalase inhibition rate amplified a relatively small molar concentration of H(2)O(2) and provided a highly specific and sensitive method for quantitatively measuring H(2)O(2). A major advantage of this approach is that it does not require extensive accumulation of H(2)O(2) in the environment. The method was successfully employed to measure H(2)O(2) secretion by Mycoplasma pneumoniae, which possesses a peroxidase-like activity that limits the accumulation of H(2)O(2) in the environment.     

Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.

Toxicity to the central nervous system (CNS) by hyperbaric oxygen (HBO) presumably relates to increased production of reactive oxygen species. The sites of generation of reactive oxygen species during HBO, however, have not been fully characterized in the brain. We investigated the relationship between regional generation of hydrogen peroxide (H2O2) in the brain in the presence of an irreversible inhibitor of catalase, aminotriazole (ATZ), and protection from CNS O2 toxicity by a monoamine oxidase (MAO) inhibitor, pargyline. At 6 ATA of oxygen, pargyline significantly protected rats from CNS O2 toxicity whereas ATZ enhanced O2 toxicity. In animals pretreated with ATZ, HBO inactivated 21-40% more catalase than air exposure in the six brain regions studied. Because ATZ-mediated inactivation of catalase was H2O2 dependent, the decrease in catalase activity during hyperoxia was proportional to the intracellular production of H2O2. Pargyline, administered 30 min before HBO, inhibited MAO by greater than 90%, prevented ATZ inhibition of catalase activity during HBO, and reversed the augmentation of CNS O2 toxicity by ATZ. These findings indicate that H2O2 generated by MAO during hyperoxia is important to the pathogenesis of CNS O2 toxicity in rats.     

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.     

Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage.

1. Activities of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) have been estimated in eye tissues. In rabbit eye, superoxide dismutase is present in corneal epithelium, corneal endothelium, lens, iris, ciliary body and retina. In lens the activity is in capsule epithelium. 2. Copper chelator diethyldithiocarbamate inhibited lens superoxide dismutase in vitro and in vivo in rabbit. 3. H2O2 caused inhibition of superoxide dismutase activity of lens extract, and this inhibition was potentiated by the catalase inhibitor 3-amino-1H-1,2,4-triazole (3-aminotriazole) or NaN3. 3-Aminotriazole or NaN3 had no effect on lens superoxide dismutase. Thus endogenous catalase of lens affords protection to the lens superoxide dismutase from inactivation by H2O2. 4. In rabbit having early cataract (vacuolar stage) induced by feeding-3-aminotriazole, there was a decrease in superoxide dismutase of lens, a fall in ascorbic acid of ocular humors and lens, and a 2--3-Fold increase in H2O2 of aqueous humor and vitreous humor. We conclude that catalase of eye affords protection to the lens from H2O2 and it also protects superoxide dismutase of lens from inactivation by H2O2. Superoxide dismutase, in turn, protects the lens from the superoxide radical, O2.-. It is likely that inhibition of these enzymes may lead to production of the highly reactive oxidant, the hydroxyl radical, under pathological conditions when H2O2 concentration in vivo exceeds physiological limits as in cataract induced by 3-aminotriazole. A scheme of reaction mechanism has been proposed to explain the relative functions of ocular catalase and superoxide dismutase. Such a mechanism may be involved in cataractogenic process in the human.     

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.     

Nitric oxide protects macrophages from hydrogen peroxide-induced apoptosis by inducing the formation of catalase

We investigated the cytoprotective effect of NO on H2O2-induced cell death in mouse macrophage-like cell line RAW264. H2O2-treated cells showed apoptotic features, such as activation of caspase-9 and caspase-3, nuclear fragmentation, and DNA fragmentation. These apoptotic features were significantly inhibited by pretreatment for 24 h with NO donors, sodium nitroprusside and 1-hydroxy-2-oxo-3,3-bis-(2-aminoethyl)-1-triazene, at a low nontoxic concentration. The cytoprotective effect of NO was abrogated by the catalase inhibitor 3-amino-1,2,4-triazole but was not affected by a glutathione synthesis inhibitor, L-buthionine-(S,R)-sulfoximine. NO donors increased the level of catalase and its activity in a concentration-dependent manner. Cycloheximide, a protein synthesis inhibitor, inhibited both the NO-induced increase in the catalase level and the cytoprotective effect of NO. These results indicate that NO at a low concentration protects macrophages from H2O2-induced apoptosis by inducing the production of catalase.     

The characteristics of the `peroxidatic' reaction of catalase in ethanol oxidation

Ethanol oxidation by rat liver catalase (the ;peroxidatic' reaction) was studied quantitatively with respect to the rate of H(2)O(2) generation, catalase haem concentration, ethanol concentration and the steady-state concentration of the catalase-H(2)O(2) intermediate (Compound I). At a low ratio of H(2)O(2)-generation rate to catalase haem concentration, the rate of ethanol oxidation was independent of the catalase haem concentration. The magnitude of the inhibition of ethanol oxidation by cyanide was not paralleled by the formation of the catalase-cyanide complex and was altered greatly by varying either the ethanol concentration or the ratio of the rate of H(2)O(2) generation to catalase haem concentration. The ethanol concentration producing a half-maximal activity was also dependent on the ratio of the H(2)O(2)-generation rate to catalase haem concentration. These phenomena are explained by changes in the proportion of the ;catalatic' and ;peroxidatic' reactions in the overall H(2)O(2)-decomposition reaction. There was a correlation between the proportion of the ;peroxidatic' reaction in the overall catalase reaction and the steady-state concentration of the catalase-H(2)O(2) intermediate. Regardless of the concentration of ethanol and the rate of H(2)O(2) generation, a half-saturation of the steady state of the catalase-H(2)O(2) intermediate indicated that about 45% of the H(2)O(2) was being utilized by the ethanol-oxidation reaction. The results reported show that the experimental results in the study on the ;microsomal ethanol-oxidation system' may be reinterpreted and the catalase ;peroxidatic' reaction provides a quantitative explanation for the activity hitherto attributed to the ;microsomal ethanol-oxidation system'.     

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.     

Effect of the in vivo catalase inhibition on aminonucleoside nephrosis.

Reactive oxygen species have been involved in the pathophysiology of puromycin aminonucleoside (PAN)-nephrosis. The role of H2O2 in these rats may be studied modulating the amount or activity of catalase, which breakdowns H2O2 to water and oxygen. To explore the role of H2O2 in this experimental model, we studied the effect of the in vivo catalase inhibiton with 3-amino-1,2,4-triazole (ATZ) on the course of PAN-nephrosis. Four groups of rats were studied: control rats (CT group), PAN-injected rats (PAN group), ATZ-injected rats (ATZ group), and ATZ- and PAN-injected rats (ATZPAN group). Rats were placed in metabolic cages to collect 24 h urine along the study, ATZ (1 g/kg) was given 24 h before PAN injection (75 mg/kg), and the proteinuria was measured on days 0, 2, 4, 6, 8, and 10. Proteinuria started before (day 4) and was significantly higher on days 6, 8, and 10 in the ATZPAN group than in the PAN group. On day 10, hypercholesterolemia was significantly higher in the ATZPAN group than in the PAN group. These data indicate that the in vivo catalase inhibition magnifies PAN-nephrosis, suggesting that H2O2 is produced in vivo and involved in the renal damage in this experimental disease.     

Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 x 10(-7) s(-1) and an endogenous H2O2 and superoxide concentrations of 5 x 10(-11) and 5 x 10(-13) M, respectively, lower than previous estimates.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

Inhibiting catalase activity sensitizes 36B10 rat glioma cells to oxidative stress

Gliomas are extremely resistant to anticancer therapies resulting in poor patient survival, due, in part, to altered expression of antioxidant enzymes. The primary antioxidant enzyme, catalase, is elevated constitutively in gliomas compared to normal astrocytes. We hypothesized that downregulating catalase in glioma cells would sensitize these cells to oxidative stress. To test this hypothesis, we implemented two approaches. The first, a pharmacological approach, used 3-amino-1,2,4-triazole, an irreversible inhibitor that reduced catalase enzymatic activity by 75%. Pharmacological inhibition of catalase was not associated with a reduction in rat 36B10 glioma cell viability until the cells were challenged with additional oxidative stress, i.e., ionizing radiation or hydrogen peroxide (H(2)O(2)). In the second molecular approach, we generated 36B10 glioma cells stably expressing catalase shRNA; a stable cell line displayed a 75% reduction in catalase immunoreactive protein and enzymatic activity. This was accompanied by an increase in intracellular reactive oxygen species and extracellular H(2)O(2). These cells exhibited increased sensitivity to radiation and H(2)O(2), which was rescued by the antioxidant, N-acetylcysteine. These results support the hypothesis that catalase is a major participant in the defense of 36B10 glioma cells against oxidative stress mediated by anticancer agents capable of increasing steady-state levels of H(2)O(2).     

Cloning and characterization of katA,encoding the major monofunctional catalase from Xanthomonas campestris pv. phaseoli and characterization of the encoded catalase KatA

The first cloning and characterization of the gene katA, encoding the major catalase (KatA), from Xanthomonas is reported. A reverse genetic approach using a synthesized katA-specific DNA probe to screen a X. campestris pv. phaseoli genomic library was employed. A positively hybridizing clone designated pKat29 that contained a full-length katA was isolated. Analysis of the nucleotide sequence revealed an open reading frame of 1,521 bp encoding a 507-amino acid protein with a theoretical molecular mass of 56 kDa. The deduced amino acid sequence of KatA revealed 84% and 78% identity to CatF of Pseudomonas syringae and KatB of P. aeruginosa, respectively. Phylogenetic analysis places Xanthomonas katA in the clade I group of bacterial catalases. Unexpectedly, expression of katA in a heterologous Escherichia coli host resulted in a temperature-sensitive expression. The KatA enzyme was purified from an overproducing mutant of X. campestris and was characterized. It has apparent K(m) and V(max) values of 75 m M [H(2)O(2)] and 2.55 x 10(5) micromol H(2)O(2) micromol heme(-1) s(-1), respectively. The enzyme is highly sensitive to 3-amino-1,2,4-triazole and NaN(3), has a narrower optimal pH range than other catalases, and is more sensitive to heat inactivation.