The three-dimensional structure of the di-Mn catalase and the environment of the di-Mn sites in different redox states

Barynin, V.V.; Hempstead, Paul D.; Vagin, A.A.; Antonyuk, S.V.; Melik-Adamyan, W.R.; Lamzin, Victor S.; Harrison, Pauline M.; Artymiuk, Peter J.

doi:10.1016/s0162-0134(97)80071-2

Cited by 102 publications

(73 citation statements)

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“…Sequence alignments with Mn-dependent catalase apoproteins from P. calidifontis VA1 (4), Salmonella enterica serovar Typhimurium (39), and L. plantarum (25) revealed that there was complete conservation of residues E 36 , E 70 , H 73 , E 155 , and H 188 , which were identified as residues that coordinate two Mn 2ϩ ions in the three-dimensional structure of the Mn-dependent catalases described so far (5)(6)(7)(8).…”

Section: Resultsmentioning

confidence: 99%

Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli

Hidalgo

Betancor

Moreno

et al. 2004

Appl Environ Microbiol

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Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H 2 O 2 -detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20-to 30-fold increase in catalase specific activity was observed, whereas a 90-to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.Catalases (EC 1.11.1.6) are widely distributed enzymes in nature that decompose hydrogen peroxide to water and molecular oxygen as a detoxifying mechanism. In addition to its crucial biological relevance, this enzyme has many industrial applications, including (i) degradation of hydrogen peroxide after textile bleaching in order to prevent problems in subsequent dyeing (12); (ii) in the dairy industry, elimination of H 2 O 2 added to sterilize milk (2, 44); (iii) coupled with oxidases, prevention of inactivation of the oxidases in the presence of high peroxide concentrations (15,23,43); and (iv) coupled with oxidases, prevention of the destruction by H 2 O 2 of the target product in an oxidase-catalyzed reaction (19,42).

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Section: Resultsmentioning

confidence: 99%

Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli

Hidalgo

Betancor

Moreno

et al. 2004

Appl Environ Microbiol

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“…4A) is half of the value (9.5 mT) observed for mononuclear Mn 2ϩ and is typical of a coupled dimanganese center in the Mn 2ϩ state. Dimanganese Mn(II) is also found in arginase and manganese catalase (22,(25)(26)(27)(28) and the hyperfine pattern observed in manganese-reconstituted C. ammoniagenes R2F resembles arginase, where the manganese center (29) is bridged by two carboxylates and one oxygen (water or hydroxyl).…”

Section: Metal Content and Epr Characteristics Of The Metal Sites-mentioning

confidence: 99%

The Active Form of the R2F Protein of Class Ib Ribonucleotide Reductase from Corynebacterium ammoniagenesIs a Diferric Protein

Huque¹,

Fieschi²,

Torrents³

et al. 2000

Journal of Biological Chemistry

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Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g ‫؍‬ 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to Xband EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.

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“…This value was comparable to the specific activities of other manganese catalases (L. plantarum, 7,800 U/mg at 25°C [9]; Thermus sp. YS 8-13, 8,000 U/mg at 65°C [20]; T. album, 17,745 U/mg at 25°C [1] Previously reported crystal structures of manganese catalases indicated the presence of two manganese atoms per subunit in the catalytic centers (3,6,7). In this study, plasma emission spectroscopy detected only 1.32 Ϯ 0.03 atoms per subunit in the P. calidifontis VA1 catalase.…”

Section: Discussionmentioning

confidence: 40%

“…Although the complete primary structure has not been published, the three-dimensional structure of T. thermophilus catalase has been determined. The structure has revealed that Glu36, Glu70, His73, Glu155 and His188 were coordinated to manganese ions in the active site (6). These residues were conserved in the P. calidifontis VA1 catalase (Fig.…”

Section: Resultsmentioning

confidence: 96%

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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The three-dimensional structure of the di-Mn catalase and the environment of the di-Mn sites in different redox states

Cited by 102 publications

References 0 publications

Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli

Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli

The Active Form of the R2F Protein of Class Ib Ribonucleotide Reductase from Corynebacterium ammoniagenesIs a Diferric Protein

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

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