Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri

Shima, Seigo; Netrusov, A. I.; Sordel, Melanie; Wicke, Michaela; Hartmann, Gudrun C.; Thauer, Rudolf K.

doi:10.1007/s002030050716

Cited by 47 publications

(33 citation statements)

References 37 publications

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“…Structure and sequence data are available for several monofunctional catalases (6,18,22). Most of these enzymes are composed of four identical protein subunits of an approximate molecular mass of 60 kDa, each containing one heme group.…”

Section: Resultsmentioning

confidence: 99%

Enterococcus faecalis Heme-Dependent Catalase

2002

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Enterococcus faecalis cells cannot synthesize porphyrins and do not rely on heme for growth but can take up heme and use it to synthesize heme proteins. We recently described a cytochrome bd in E. faecalis strain V583 and here report the identification of a chromosomal gene, katA, encoding a heme-containing cytoplasmic catalase. The 54-kDa KatA polypeptide shows sequence similarity to members of the family of monofunctional catalases. A hexahistidyl-tagged version of the catalase was purified, and major characteristics of the enzyme were determined. It contains one protoheme IX group per KatA polypeptide. Catalase activity was detected only in E. faecalis cells grown in the presence of heme in the medium; about 2 and 10 M hemin was required for half-maximal and maximal production of catalase, respectively. Our finding of a catalase whose synthesis is dependent on the acquisition of heme in the opportunistic pathogen E. faecalis might be of clinical importance. Studies of cellular heme transport and heme protein assembly and in vivo synthesis of metalloprotein analogs for biotechnological applications are impeded by the lack of experimental systems. We conclude that the E. faecalis cell potentially provides such a desired system.Enterococcus faecalis, formerly known as Streptococcus faecalis, is a gram-positive bacterium with a low GϩC content in its genomic DNA. It is a common inhabitant of the intestines of animals, including humans, where it is part of the commensal flora. However, E. faecalis can cause disease in, e.g., immunodeficient persons, and it is frequently the causative agent of nosocomial infections. Lately, the emergence of multidrugresistant strains has become a serious problem (10). Recognizing the medical importance of this bacterium, The Institute for Genomic Research has undertaken the genome sequencing of E. faecalis strain V583.Heme is present as a prosthetic group in a variety of proteins, including, for example, catalases, cytochromes, and hemoglobins. E. faecalis cells do not synthesize heme, and genes for known porphyrin biosynthetic enzymes are not found in the genome sequence of strain V583. However, if E. faecalis cells are supplied with heme, synthesis of hemoproteins can take place (16,19). We recently described a functional cytochrome bd-type quinol oxidase in E. faecalis V583 (27). The cytochrome bd is probably the terminal oxidase of a respiratory chain present in E. faecalis under certain conditions (1, 15). The presence of an aerobic respiratory chain is puzzling, however, in a bacterium generally considered to use a fermentative-energy metabolism. Aerobic respiration is more energyefficient than fermentation, but it is also a source of reactive oxygen species. Cells have several protective mechanisms against these toxic compounds. Enzymatic detoxification of hydrogen peroxide in bacteria is mainly performed by catalases. Three classes of bacterial catalases have been described: monofunctional catalases, catalase-peroxidases, and manganese catalases (pseudocatalases) (30). The ...

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Section: Resultsmentioning

confidence: 99%

Enterococcus faecalis Heme-Dependent Catalase

2002

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“…The (14), and M. barkeri (34). Conserved amino acids that form part of the active (*) and heme coordination (͉) sites are indicated.…”

Section: Resultsmentioning

confidence: 99%

“…There are three separate families of catalases: Mn-catalases, bifunctional catalase-peroxidases, and monofunctional, or "true," catalases. The last group is the one best characterized and corresponds to homotetrameric heme-containing enzymes present in eubacteria and eukaryotes and recently also found in the Archaea (34). Within this family of catalases, two clearly distinct classes can be recognized: the small-subunit (50-to 65-kDa) and the large-subunit (ϳ80-kDa) enzymes.…”

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confidence: 99%

Multiple Catalase Genes Are Differentially Regulated in Aspergillus nidulans

2001

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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 (ϳ84 kDa) and CatB (ϳ79 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.Several studies indicate that reactive oxygen species play crucial roles in various aspects of cell physiology, such as cellular defense (45), life span (38), stress signaling (22), development (19), apoptosis (30), and pathology (33). The hydrogen peroxide formed during aerobic metabolism is capable of generating other reactive oxygen species, which can damage many cellular components (18). Catalases and peroxidases are the most important enzymatic systems used to degrade H 2 O 2 . There are three separate families of catalases: Mn-catalases, bifunctional catalase-peroxidases, and monofunctional, or "true," catalases. The last group is the one best characterized and corresponds to homotetrameric heme-containing enzymes present in eubacteria and eukaryotes and recently also found in the Archaea (34). Within this family of catalases, two clearly distinct classes can be recognized: the small-subunit (50-to 65-kDa) and the large-subunit (ϳ80-kDa) enzymes. The first class includes a large number of catalases from bacteria, plants, fungi, and animals. An increasing number of catalases of the second class have been identified in bacteria and filamentous fungi (5,8,13,15,(23)(24)(25)27, 37) but not in higher eukaryotes.The core sequence of the true catalases is composed of 360 to 390 amino acid resid...

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“…(11,12,14,41,42), but there have been no previous reports of a manganese catalase. Recently, an abundant number of complete genome sequences from various archaeal strains have been determined.…”

Section: Discussionmentioning

confidence: 99%

“…A catalase-peroxidase and a monofunctional catalase were purified from the halophilic archaeon Halobacterium halobium (11,12), and a catalase-peroxidase was also purified from Haloarcula marismortui (14). In methanogenic archaea, monofunctional catalases were purified from Methanosarcina barkeri (41) and Methanobrevibacter arboriphilus (42). In (hyper)thermophilic archaea, a putative catalase-peroxidase gene was found in the genome of the obligately anaerobic archaeon Archaeoglobus fulgidus (26).…”

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confidence: 99%

Unique Presence of a Manganese Catalase in a Hyperthermophilic Archaeon, Pyrobaculum calidifontis VA1

2002

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We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70°C. The enzyme exhibited a K m value of 170 mM toward H 2 O 2 and a k cat value of 2.9 ؋ 10 4 s ؊1 ·subunit ؊1at 25°C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat Pc ). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat Pc revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)-thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat Pc can be considered a rare example of a manganese catalase from archaea.In aerobic organisms, hydrogen peroxide is produced by various enzymatic reactions, particularly the univalent reduction of molecular oxygen by oxidases and the disproportionation of superoxide by superoxide dismutases. Hydrogen peroxide is a toxic molecule as it can both oxidize and reduce organic substrates in cells. Catalases (EC 1.11.1.6) remove this toxicity by catalyzing the disproportionation of hydrogen peroxide into molecular oxygen and water. Along with superoxide dismutases, catalases play an important role in defending the cell against oxidative stress, and are distributed in almost all aerobic and facultatively anaerobic organisms.Catalases are assigned to three phylogenetically distinct groups: two groups comprised of heme catalases and one group of nonheme catalases (29). The monofunctional (or typical) catalases, which are one type of heme catalases, have been found in many bacteria, archaea, plants, fungi, and animals. They display a broad range of subunit sizes (55 to 84 kDa) and are generally tetrameric enzymes. Monofuncti...

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Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri

Cited by 47 publications

References 37 publications

Enterococcus faecalis Heme-Dependent Catalase

Enterococcus faecalis Heme-Dependent Catalase

Multiple Catalase Genes Are Differentially Regulated in Aspergillus nidulans

Unique Presence of a Manganese Catalase in a Hyperthermophilic Archaeon, Pyrobaculum calidifontis VA1

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